0013
c ^"
January 1992
FINAL
DRINKING WATER CRITERIA DOCUMENT
FOR
^ ENDOTHALL
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460
HEADQUARTERS LIBRARY
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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January 1992
FINAL
DRINKING WATER CRITERIA DOCUMENT
FOR
ENDOTHALL
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460
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TABLE OF CONTENTS
Page
LIST OF TABLES v
FOREWORD vi
AUTHORS, CONTRIBUTORS, AND REVIEWERS viii
I. SUMMARY [-1
II. PHYSICAL AND CHEMICAL PROPERTIES II-I
A. General Properties II-l
B. Manufacture and Use II-l
C. Environmental Fate and Stability ........ Il-i
D. Summary . II-4
III. TOXICOKINETICS III-l
A. Absorption III-l
B. Distribution III-l
C. Metabolism III-6
D. Excretion III-6
E. Summary II1-8
IV. HUMAN EXPOSURE IV-1
A. Exposure Estimation IV-2
1. Drinking Water IV-1
2. Diet IV-2
3. Air IV-3
B. Summary IV-3
V. HEALTH EFFECTS IN ANIMALS V-l
A. Short-term Exposure V-l
I. Lethality V-l
2. Other Effects . V-l
B. Long-term Exposure . V-5
C. Reproductive/Teratogenic Effects V-10
D. Mutagenicity . . . V-12
1. Gene Mutation Assays (Category 1) ..-....• V-15
2. Chromosome Aberration Assays (Category 2) V-18
3. Other Mutagenic Mechanisms (Category 3) V-19
iii
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TABLE OF CONTENTS (continued)
Pace
E. Careinogenicity V-24
F. Summary V-24
VI. HEALTH EFFECTS IN HUMANS VI-1
A. Clinical Case Reports VI-I
B. Epidemic logical Studies VI-1
C. High-Risk Groups VI-1
D. Summary ' vi-1
VII. MECHANISMS OF TOXICITY VII-1
A. In Animals . . . . VII-1
B. In Plants VII-1
C. Synergistic/Antagonistic Effects . VII-1
D. Summary VI1-2
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS VIII-1
A. Procedures for Quantification of Toxicological Effects . VIII-1
1. Noncarcinogenic Effects VIII-1 a
2. Carcinogenic Effects VIII-4 'I
B. Quantification of Noncarcinogenic Effects for Endothall . VIII-6
1. One-day Health Advisory VIII-6
2. Ten-day Health Advisory VIII-6
3. Longer-term Health Advisory VIII-8
4. Reference Dose and Drinking Water Equivalent Level . VUI-9
C. Quantification of Carcinogenic Effects for Endothall . . VIII-11
1. Categorization of Carcinogenic Potential VIII-11
2. Quantitative Carcinogenic Risk Estimates VIII-12
D. Summary VIII-12
IX. REFERENCES IX-1
iv
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LIST OF TABLES
Table No. paae
. II-l Properties of En'dothall (7-oxabicyclo-(2.2.1)-heptane-
2,3-dicarboxylic acid) ' II-2
III-l Percent of Radioactive Dose in Tissues of Rats
Receiving an Oral Dose of "(^Labeled Endothall and
Sacrificed at Various Times After Dosing III-3
III-2 Radioactivity in Tissues of Rats Receiving an Oral Dose
of 14C-Labeled Endothall and Sacrificed at Various
Times After Dosing . r- III-4
III-3 Kinetics of Radioactivity Elimination After a Single
Oral Dose of 1 mg of 14C-Endothall per Rat . • . 111-5
III-4 Excretion of Radioactivity by Rats Receiving a Single
Oral Dose of 14C Ring-Labeled Endothall III-7
V-l Summary of Oral Lethality Data on Endothall in Rats ... V-2
V-2 Acute Inhalation Toxicity of Endothall and Formulations
(Aqueous Aerosols) ... V-4
V-3 Primary Dermal Irritation of Endothall and Formulations
in Rabbits V-6
V-4 Primary Eye Irritation of Endothall and Formulations
in Rabbits V-7
V-5 Summary of Genotoxicity Data on Endothall V-13
V-6 Effect of Aquathol K on Transformation of BALB/3T3
Cells Without Metabolic Activation V-21
V-7 Effect of Aquathol K on Transformation of BALB/3T3
Cells in the Presence of Primary Rat Hepatocytes .... V-22
VIII-1 Summary of Candidate Studies for Derivation of the
Ten-day Health Advisory for Endothall VIII-7
VIII-2 Summary of Candidate Studies for Derivation of the DUEL
for Endothall .. VIII-10
VIII-3 Summary of Quantification of Toxicological Effects
for Endothall • VIII-13
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as
amended in 1986, requires the Administrator of the Environmental
Protection Agency to publish Maximum Contaminant Level Goals
(MCLGs) and promulgate National Primary Drinking Water Regulations
for each contaminant, which, in the judgment of the Administrator,
may have an adverse effect on public health and which is known or
anticipated to occur in public water systems. The MCLG is
nonenforceable and is set at a level at which no known or
anticipated adverse health effects in humans occur and which allows
for an adequate margin of safety. Factors considered in setting
the MCLG include health effects data and sources of exposure other
than drinking water.
This document provides the health effects basis to be
considered in establishing the MCLG. To achieve this objective,
data on pharmacokinetics, human exposure, acute and chronic
toxicity to animals and humans, epidemiology, and mechanisms of
toxicity were evaluated. Specific emphasis is placed on literature
data providing dose-response information. Thus, while the
literature search and evaluation performed in support of this
document was comprehensive, only the reports considered most
pertinent in the derivation of the MCLG are cited in the document.
The comprehensive literature data base in support of this document
includes information published up to April 1987; however, more
recent data may have been added during the review process..
When adequate health effects data exist. Health Advisory values
for lessthan-lifetime exposures (One-day, Ten-day, and Longer-term,
approximately 10% of an individual's lifetime) are included in this
document. These values are not used in setting the MCLG, but serve
as informal guidance to municipalities and other organizations when
emergency spills or contamination situations occur.
James R. Elder
Director
Office of Ground Water and Drinking Water
Tudor T. Davies
Director
Office of science and Technology
vi
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended In 1986,
requires the Administrator of the Environmental Protection Agency to publish
Maximum Contaminant Level Goals (MCLGs) and promulgate National Primary Drinking
Water Regulations for each contaminant, which, in the judgment of the
Administrator, may have an adverse effect on public health and which is known or
anticipated to occur in public water systems. The MCLG is nonenforceable and is
set at a level at which no known or anticipated adverse health effects in humans
occur and which allows for an adequate margin of safety. Factors considered in
setting the MCLG include health effects data and sources of exposure other than
drinking water.
This document provides the health effects basis to be considered in
establishing the MCLG. To achieve this objective, data on pharmacokinetics,
human exposure, acute and chronic toxicity to animals and humans,-epidemiology,
and mechanisms of toxicity were evaluated. Specific emphasis is placed on
literature data providing dose-response information. Thus, while the literature
search and evaluation performed in support of this document was comprehensive,
only the reports considered most pertinent in the derivation of the MCLG are
cited in the document. The comprehensive literature data base in support of this
document includes information published up to April 1987; however, more recent
data have been added during the review process and in response to public
comments.
When adequate health effects data exist, Health Advisory values for less-
than-lifetime exposures (One-day, Ten-day, and Longer-term, approximately 10% of
an individual's lifetime) are included in this document. These values are not
used in setting the MCLG, but serve as informal guidance to municipalities and
other organizations when emergency spills or contamination situations occur.
James R. Elder
Director
Office of Ground Water and Drinking Water
Tudor T. Davies
Director
Office of Science of Technology
vi
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
The following Dynamac Corporation personnel were involved In the preparation
of this document: Nicolas P. Hajjar, Ph.D. (Department Manager); Norbert Page,
D.V.M. (Technical Director); Alberto Pretzel, Ph.D. (Principal Author); Nancy
McCarroll 8.S., and Michael Narotsky (Authors); Karen Swetlow, B.S. (Technical
Editor); William McLellan, Ph.D. (Reviewer); Gloria Fine and Sanjivani Diwan,
Ph.D. (Information Specialists).
This document was prepared under a contract to Environmental Management
Support, Inc., with the Criteria and Standards Division, Office of Drinking
Water, U.S. Environmental Protection Agency (U.S. EPA), Washington, DC (Robert
Cantilli, Lead Scientist and Contract Manager).
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I. SUMMARY
Endothall is an organic acid (7-oxabicyclo-(2.2.1)-heptane-2,3-dicar-
boxylic acid). It is soluble in .water, methanol, and acetone and to some
extent in other solvents. This dicarboxylic acid loses water at 90°C to yield
the corresponding acid anhydride, a powerful vesicant.
Endothall salts are widely used as defoliants and as herbicides for con-
trol of terrestrial and aquatic weeds. Because of its water solubility,
endothall tends to follow water movement in the environment. In ponds and
lakes, the compound disappears from the water and the hydrosoil within 30 to
60 days and, often, within a considerably shorter period. This appears to be
due primarily to microbial degradation of endothall.
One study was found in the available literature that provides informa-
tion, although limited, on the absorption, distribution, metabolism, and
excretion of endothall in mammals. In rats administered a single oral dose of
about 5 ing/kg "C ring-labeled (at C, and C2) endothall, approximately 3% of
the dose was expired as carbon dioxide, 7% was excreted in the urine, and the
remainder (90%) appeared in the feces. Total recovery was 95 to 99% within
48 hours. Most of the compound is excreted in the feces as the unchanged
chemical, suggesting that there is little gastrointestinal absorption.
Endothall was also the only compound detected in the urine.
Absorbed endothall distributes widely throughout the body tissues. In
rats that received a single oral dose of 1.0 mg 14C-endothall, the highest "C
levels observed I hour after dosing were in stomach and intestines (about 95%)
liver (1.1%) and kidney (0.9%) tissues, with lower amounts (0.02 to 0.1%) in
heart, lung, spleen, and brain. Very low 14C levels were observed in muscle
and fat. Tissue residues fell to unmeasurable levels within 48 to 72 hours;
therefore, endothall would not be expected to accumulate.
1-1
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Data on the Intake of endothall from drinking water, food, and ambient
air are insufficient for use in determining which of the three sources is the
major contributor to total intake.
Estimates of the acute oral LD,0 values for endothall ion in various
endothall formulations In rats range from 31 to 138 mg/kg. Single intravenous
doses of 5 mg endothall/kg or higher were fatal to dogs and rabbits. Death
was attributed to either respiratory or cardiac failure. Inhalation exposure
at levels above 3,000 mg endothall ion/m1 for 1 hour resulted in lung
congestion and hemorrhage in exposed rats. Endothall is irritating to the
skin and corrosive to the eyes of rabbits.
Rats that received oral doses of approximately 400 mg endothall ion/kg
body weight (bw)/day died within 1 week. Necropsy examination revealed slight
liver degeneration and focal hemorrhagic areas in the kidneys.
A 2-year toxicity study with disodium endothall in dogs provided a No-
Observed-Adyerse-Effect Level (NOAEL) of 2 mg endothall ion/kg/day and a
Lowest-Observed-Adverse-Effect Level (LOAEL) of 6 mg endothall ion/kg/day
based on increased organ weights and organ-to-body weight ratios for the
stomach and small intestine.
In a similar 1-year toxicity study in dogs, hyperplastic changes in the
portal tract of the liver and dose-related reactive hyperplastic changes in
the mucosa of the stomach were observed at 14.4 mg/kg/day; at 4.8 mg/kg
endothall ion/day, no effects were observed in the liver, and marginal injury
to the stomach was observed.
A 2-year toxicity study with disodium endothall in rats, with small
numbers of animals in control and treated groups, suggests a NOAEL of 100 mg
endothall ion/kg/day, the highest dose tested. No toxic effects other than
lethality were reported.
1-2
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A three-generation reproduction study with disodium endothall in rats
demonstrated toxic effects and increased mortality in the offspring at 12 mg
endothall 1on/kg/day in the maternal diet. At 4 rag endothall ion/kg/day, no
effects were observed in either the dams or the offspring. Although maternal
and fetal toxicity was evident at high-dose levels, no abnormalities were
evident in the offspring in any generation. Thus, a NOAEL of 4 mg endothall
ion/kg/day and a LOAEL of 12 mg endothall ion/kg/day were established for
developmental/reproductive toxicity.
A teratology study 1n rats indicated that endothall (technical 89.5% acid
equivalent) is neither embryo-toxic nor teratogenlc at maternal doses of
30 mg/kg/day or less during gestation days 6 to 19; however, maternal toxicity
was observed at 20 and 30 mg endothall/kg/day. This study provided a NOAEL of
30 mg/kg/day for developmental toxicity, and a NOAEL and LOAEL of 10 and
20 mg/kg/day, respectively, for maternal toxicity. A similar study in mice
indicated that endothall (technical 89.5% acid equivalent) was teratogenlc at
maternal doses of 40 mg/kg/day; however, maternal toxicity also occurred at
this dose and at 20 mg/kg/day. This study provided a NOAEL and a LOAEL for
maternal toxicity of 5 and 20 mg endothall technicalAg/day, respectively, and
a NOAEL and a LOAEL for developmental toxicity of 20 and 40 mg endothall
technical/kg/day, respectively.
Endothall was not mutagenic in bacterial, fungal, mammalian cell, or
Drosophila assays. In in vivo somatic or male germinal cell cytogenetic
assays, within the nontoxic dose ranges, no clastogenic effects were observed.
Endothall did not induce aneuploidy in plants or increase the frequency of
sister chromatid exchanges in human lymphocytes. Although Aquatol K\ (dipo-
tassium endothall) exhibited transforming activity in BALB/3T3 cells co-
cultivated with primary rat hepatocytes, the results are questionable because
of weaknesses in the study.
Although endothall has not been shown to be carcinogenic, the present
data base is inadequate to assess its carcinogenic potential.
1-3
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One case report of endothall poisoning In humans was found in the avail-
able literature. This involved the suicide of a young man who ingested an
estimated 7 to 8 g of endothall (approximately 100 mg endothall ion/kg) in a
solution containing 175 g/L. Repeated vomiting, which occurred after inges-
tion, evidently removed much of the compound and may have resulted in aspira-
tion of the compound to the lung. The autopsy report revealed gross hemor-
rhage in the gastrointestinal tract and widespread focal hemorrhages and edema
of the lungs. No epidemiological studies of endothall exposure have been
reported. The mechanism of endothall toxicity in humans is not known.
No specific information is available on the mechanism of toxicity of
endothall in animals. Intravenous toxicity studies in dogs suggest that the
cause of death is cardiac or respiratory arrest. One study suggests that
isopropylphenylcarbamate may antagonize endothall toxicity, but data were not
provided.
Available data from short-term oral toxicity studies are essentially
restricted to mortality data and are not suitable for Health Advisory (HA)
calculations. A 10-day teratology study provided a NOAEL of 8 mg endothall
ion/kg/day based on a lack of maternal effects and effects in offspring.
Using this NOAEL, a Ten-day HA for children was calculated to be 800 »g/L.
Since no suitable data are available for calculation of a One-day HA, the Ten-
day HA value may be used as a conservative estimate of the One-day HA. There
were insufficient data for calculation of the Longer-term HA values. There-
fore, the DWEL adjusted for a 10-kg child (200.^g/L) may be used to estimate
the Longer-term HA for a 10-kg child; and the DWEL (700 »g/L) may be used to
estimate the Longer-term HA for a 70-kg adult. Using a NOAEL of 2.0 mg
endothall ion/kg/day from a 2-year study in dogs, a Reference Dose (RfD) of
20 ng endothall ion/kg/ day and a Drinking Water Equivalent Level (DWEL) of
700 »g/L were calculated. No estimations of excess cancer risk were
performed.
1-4
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There are no existing guidelines or exposure standards available for
endothall. Residue tolerances of 0.05 to 0.1 ppm on crops have been estab-
lished. An Interim tolerance of 200 »g/L has been published for residues of
endothall, used to control aquatic plants, 1n potable water.
1-5
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II. PHYSICAL AND CHEMICAL PROPERTIES
A. GENERAL PROPERTIES
Endothall is an organic acid (7-oxabicyclo-(2.2.1)-heptane-2,3-dicar-
boxylic acid) that is soluble in water, methanol, and acetone and to some
extent in other solvents (Carlson et al., 1978). Its physical and chemical
properties are summarized in Table II-l. Endothall loses water at 90°C to
yield the corresponding acid anhydride, which is a powerful vesicant and eye
irritant (Carlson et al., 1978; Windholz et al., 1983). Endothall monohydrate
melts at 144°C, with loss of water, to yield the corresponding acid anhydride
(Carlson et al., 1978).
B. MANUFACTURE AND USE
Endothall is utilized as a defoliant for a wide range of crops and as a
herbicide for both terrestrial and aquatic weeds. Endothall is prepared by
the Diels-Alder addition of maleic anhydride to furan to yield the
dicarboxylic acid (Carlson et al., 1978). Commercial formulations generally
contain the sodium, potassium, or N,N-dialkylamine salts of the acid (Simsiman
et al., 1976).
C. ENVIRONMENTAL FATE AND STABILITY
Since endothall salts are water soluble, endothall tends to move with
water through soil. Soil texture and organic content influence the rapidity
of its movement (Simsiman et al., 1976). Yeo (1970) reported on the
disappearance of endothall from farm reservoirs. The water temperature at a
depth of 30 cm in the reservoirs ranged from 16.5* to 27.5°C, and the pH
ranged from 7.2 to 9.1. In 4 of 14 applications to the farm reservoirs, the
concentration of endothall decreased from initial values of 0.3 to 1.4 mg/L to
near the limit of detection within 8 to 20 days. In 10 of 14 applications to
the reservoirs, the decrease was slower, with an average of 71% of the initial
endothall disappearing in the first 12 days.
II-l
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Table II-l. Properties of Endothall (7-oxabicyclo-(2.2.1)-
heptane-2,3-dicarboxylic acfd)
m
Property
Value
CAS No.
RTECS Mo.
Synonyms
Molecular formula
Molecular weight
Physical appearance
Melting point
Density
Vapor pressure
pK, (20°C)
pK, (20eC)
Solubility:
Acetone.
Benzene
Dioxane
Ether
Isopropyl alcohol
Methanol
Water
145-73-3
RN7875000
Hexahvdro-.3.6-endo-oxy-Dhthalic
acid; 3,6-gndfi-epoxy-l,2-cyclo-
hexanedicarboxylic acid
C,H1005
186.06
White, crystalline solid
Converts to the anhydride at 90°C"
1.43 g/mL
Negligible
3.4
6.7
g acid monohydrate/100 g solvent:
7.0
0.01
7.6
0.1
1.7
28.0
10.0
•From Windholz et al. (1983).
"Endothall monohydrate melts at 144°C, with formation of the anhydride
(Carlson et al., 1978).
SOURCE: Adapted from Carlson et al. (1978) and Simsiraan et al. (1976), except
where indicated.
II-2
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Sikka and Rice (1973J reported that in a pond treated with 2 mg/L
endothall, the compound was undetectable within 36 days in the water and
within 44 days in the hydrosoil (silt-loam). In aquaria studies utilizing the
same pond water and hydrosoil, end.othall (2 to 4 mg/L initially) was undetect-
able (<0.01 mg/L) by the end of 7 days. Levels in the hydrosoil fell to below
0.1 mg/L within 2 to 4 weeks. When the water and soil were sterilized (by
autoclaving), endothall showed an initial transport into the hydrosoil, after
which the total system then remained relatively stable. This indicated that
the loss of endothall from the nonautoclaved pond water and soil was largely
due to microbial degradation. Sikka and Saxena (1973) reported that aquatic
microorganisms readily degrade endothall, and that one species of Arthrobacter
could utilize endothall as the sole carbon source. This organism incorporated
14C from ring-labeled (at C2 and Cj) endothall into cellular amino acids,
proteins, nucleic acids, and lipids, and released '*C as carbon dioxide.
After examining the pattern of label incorporation into intermediary
metabolites, the authors concluded that the 14C from ring-labeled endothall
was incorporated into glutamic acid via the tricarboxylic acid cycle and by an
alternative, unknown pathway.
Sikka and Rice (1973) reported that endothall disappeared from water in
three phases: the initial rapid rate of disappearance was attributed to
adsorption by the hydrosoil; a second, considerably slower rate of disap-
pearance was attributed to microbial metabolism; and a third, intermediate
rate of disappearance was suggested to be due to the proliferation of micro-
organisms with the ability to degrade endothall. Simsiman and Chesters (1975)
reported that in a simulated lake impoundment, 72% of the added endothall
(3 mg/L) persisted for more than 30 days because of prolonged oxygen depletion
following weedkill. A more rapid degradation of endothall occurred after the
restoration of oxygenated conditions; endothall was not detectable after
60 days. Thus, although the rate of disappearance of endothall in the
environment may vary considerably depending on conditions, it is usually
cleared from soil and water within 30 to 60 days.
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Levels of endothall in drinking water and their relationship to human
exposure are reviewed In Chapter IV of this document.
0. SUMMARY
Endothall, a water-soluble dibasic organic acid, is used primarily in the
form of its sodium, potassium, or alkylamine salts as a terrestrial and
aquatic herbicide. Because of Its water solubility and its relatively rapid
degradation rate, it is widely used to kill aquatic weeds. Natural pond and
aquaria studies demonstrate that endothall is rapidly decomposed by soil and
aquatic microorganisms and has a relatively short half-life in the
environment.
II-4
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III. TOXICOKINETICS
A. ABSORPTION
A study conducted by Soo et al. (1967) in rats provides most of the
information on the absorption, distribution, metabolism, and excretion of
endothall in orally dosed laboratory animals. Each of six Wistar rats
(3 months old; two males, four females) was administered, by gavage, 1 rag of
14C ring-labeled endothall (approximately 3.9 mg endothall/kg for males and
5.2 mg endothall/kg for females). The compound was radiolabeled in carbons 1
and 2 of the oxabicyclo ring. For dosing, the endothall was dissolved in 1 ml
of 20% ethanol. For at least 2 weeks prior to dosing, the rats received
unlabeled endothall in their feed at a concentration of 5 mg endothall/kg feed
(corresponding to approximately 0.254 mg endothall/kg bw/day for males and
0.335 mg endothall/kg bw/day for females). There were no observed signs of
toxicity. Between 85 and 91% of the radioactive dose was recovered in the
feces as untransformed endothall. Urinary excretion accounted for approxi-
mately 7% of the dose; approximately 3% of the radioactivity was recovered in
expired carbon dioxide (see also Section III.D, Excretion). The results
suggested that only about 10% of the oral dose is absorbed in rats. It is not
clear to what extent the use of 20% ethanol as a dosing vehicle may have
influenced the extent of endothall absorption and metabolism.
B. DISTRIBUTION
Soo et al. (1967) reported on the tissue distribution of WC in Wistar
rats after they were administered an oral dose (by gavage) of 1 mg of MC
ring-labeled compound (approximately 5.2 mg endothall/kg). Nine female rats
(3 months old) were maintained on a regular laboratory diet containing
unlabeled endothall at a concentration of 5 mg endothall/kg diet (approxi-
mately 0.335 mg endothall/kg bw/day) for at least 2 weeks prior to the
administration of the radioactive endothal.l. Food and water were available
ad libitum during the study period. After receiving the oral dose of labeled
III-l
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endothall, the rats were sacrificed at intervals ranging from I to 72 hours
for analysis of radioactivity in tissues.
The results of this study are shown in Tables III-l and III-2 (Soo
et al., 1967). Peak "C levels in all tissues were observed 1 hour after
dosing. Most of the dose (about 95%) was present in the stomach and
intestine, with the next highest levels in liver and kidney. Very low levels
were observed in muscle, and levels in fat were, too low to measure. During
the first 4 hours, "C levels in the stomach decreased, while levels in the
intestines increased. After 72 hours, all tissue levels had returned to zero.
Only a trace of the compound remained in the Intestinal tract.
Calculations by the authors (Table III-3) indicated a half-life for
endothall in the intestine and liver of 14.4 and 21.6 hours, respectively.
Disappearance from the stomach was biphasic, with a rapid half-life of 2.2
hours and a slower half-life of 14.4 hours. The kidney also showed a biphasic
clearance, with an initial half-life of 1.6 hours and a slower half-life of
34.6 hours.
Soo et al. (1967) also fed endothall to two lactating rats to determine
whether endothall was secreted in milk. The animals received daily oral doses
of 0.2 mg endothall (nonradioactive, in a 10% sucrose solution) for 1 week
prior to parturition. After birth, the dams received a daily dose of 0.4 mg
of 14Cendothall, in a 10% sucrose solution, for 5 consecutive days. Tissues
and stomach contents from the pups exhibited no radioactivity, suggesting that
endothall was not secreted in the milk of lactating rats.
C. METABOLISM
Soo et al. (1967) studied the excretion of endothall in the urine and
feces of two male and four female Wistar rats (3 months old) that were
administered a single oral dose (by stomach tube) of 5 mg 14C-endothall/kg.
Endothall was the only compound detected in urine by paper chromatography
using different solvent systems.
III-2
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Table III-l.
Percent of Radioactive Dose in Tissues of Rats Receiving
an Oral Dose of 14C-Labeled Endothall* and Sacrificed at
Various Times After Dosing
Time of sacrifice
Tissue
1
2
• 4
oostdosina
6
(hours):
8
12
Liver
Kidney
Heart
Lung
Spleen
Brain
Stomach
Intestine
13
86
05
11
02
03
75.18
23.59
60
26
02
04
01
02
68.80
28.75
56
27
02
04
0.01
0.02
40.29
65.85
0.49
0.07
.02
.02
0.01
0.01
9.46
61.43
0.
0.
,32
,06
,01
,02
,01
,01
,88
54.05
0.31
0.06
0.02
0.03
0.01
0.00
0.25
15.97
'Specific activity - 407 counts per minute (cpm)/ng endothall. Total dose
1.0 mg/rat (407,000 cpm), equivalent to about 5 mg/kg.
SOURCE: Adapted from Soo et al. (1967).
III-3
-------
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o» o o
*J O v>
IV
u
U
III-4
-------
Table III-3. Kinetics of Radioactivity Elimination After a Single OralDose
of 1 rag of 14C-Endothall per Rat*
Tissue k" • t,,7e
Intestine 0.048 14.4
Stomach
Rapid phase 0.316 2.2
Slow phase 0.048 14.4
Kidney
Rapid phase 0.446 1.6
Slow phase 0.020 34.6
Liver' 0.032 21.6
•Rats received a single oral dose of 1 mg of "C-endothall, equivalent to a
dose of about 5 mg/kg bw.
"Elimination rate constant.
eHalf-life (hours).
"Similar numbers for spleen, brain, lung, and heart (numerical values not
reported).
SOURCE: Adapted from Soo et al. (1967).
III-5
-------
Unchanged endothall has also been found in exposed fish. Sikka et al.
(1975) studied the metabolism of endothall in the bluegill. This study was
limited to chromatography of an extract of whole fish after the animals were
exposed for 48 hours to 2 mg 14C-endothall/L. Whole, homogenized fish were
extracted with methanol, and the extract was concentrated under vacuum. The
aqueous residue was extracted twice with petroleum ether to remove lipids.
The aqueous phase contained essentially all of the radioactivity, suggesting
that no lipid-soluble metabolites were formed. .Thin-layer chromatography on
silica gel and cellulose plates revealed the presence, of only unchanged
endothall. Thus, the methodology used by the authors revealed no metabolites
of endothall in whole fish.
0. EXCRETION
Soo et al. (1967) investigated the excretion of endothall in orally dosed
laboratory animals (rats). The results are summarized in Table III-4. Within
48 hours, nearly all of the label could be accounted for by excretion in the
feces, urine, and expired air. . From 2.5 to 2.8% of the total dose was
excreted as carbon dioxide (all in the first 24 hours). About 6 to 7%
appeared in the urine, and the remainder (85 to 91%) appeared in the feces.
Results of this study suggest that no significant bioaccumulation or retention
occurs.
E. SUMMARY
Following a single oral dose of 1 mg (about 5.2 mg/kg) endothall in rats,
about 10% of the dose was absorbed. Most of the dose was excreted in the
feces as untransformed endothall, while 7% appeared in the urine and 3% was
eliminated as expired CO,. Endothall that was absorbed was distributed in low
levels through most tissues of the body. The tissue with the highest levels
(cpm/100 mg dry tissue) at 1 hour was the kidney, but no marked preferential
accumulation was apparent. Clearance of endothall was biphasic in stomach
(t,,, » 2.2 and 14.2 hours) and kidney.(t1/2 » 1.6 and 34.6 hours),
in-6
-------
Table III-4. Excretion of Radioactivity by Rats Receiving a
Single Oral Dose* of "C Ring-Labeled Endothall
RecoveYv of UC label (X total)
Rat
1
2
3
4
S
6"
Sex
F
H
F
N
F
f
Carbon
Body
weight
(9)
193
250
206
261
206
172
dioxide Urine
Day
1
2.8
2.6
2.S
2.5
2.6
2.6
Day
2
0
0
0
0
0
Day
1
6.7
6.8
5.9
5.3
S.6
3.4
Day
2
0.4
0.5
0.6
0.4
0.3
64.5
Feces
Day
1
71.2
67.1
68.3
67.3
70.6
Day
2
17.7
17.6
20.7
20.1
20.5
48-hour
recovery
(X)
98.8
94.6
98.0
95.6
99.6
72 -hour
recovery
(X)
99.1
94.8
98.5
95. 7s-'
99.6
'Total dose » 1 mg/rat, equivalent to about 5 mg/kg.
"Rat No. S sacrificed 48 hours after dosing.
'Total recovery for rat No. 5 given as 97X in original reference; however, actual total
was 99.6X. as shown above.
"Rat No. 6 sacrificed 24 hours after dosing.
SOURCE: Adapted from Soo et al. (1967).
III-7
-------
and monophasic in intestine and liver (t,,, - 14.4 and 21.6 hours, respec-
tively). Total excretion was greater than 95% of the dose by 48 hours and
greater than 99% by 72 hours, suggesting that no significant bioaccuimilation
"or retention occurs.
i
III-8
-------
IV. HUMAN EXPOSURE
Humans may be exposed to chemicals such as endothall from a variety of
sources, including drinking water,-food, ambient air, occupational settings,
and consumer products. This analysis of human exposure to endothall is
limited to drinking water, food, and ambient air because those media are
considered to be sources common to all individuals. Even in limiting the
analysis to these three sources, it must be recognized that individual
exposure will vary widely based on many personal choices and on several
factors over which little control exists. Where one lives, works, and
travels, what one eats, and physiologic characteristics related to age, sex,
and health status can all profoundly affect daily exposure and intake.
Individuals living in the same neighborhood or even in the same household can
experience vastly different exposure patterns.
Information concerning the occurrence of and exposure to endothall in the
environment is presented in another document entitled "Occurrence of
Pesticides in Drinking Water, Food, and Air" (Johnston et al., 1984). This
chapter summarizes the pertinent information presented in that document in
order to assess the relative source contribution from drinking water, food,
and air.
In the Exposure Estimation section of this chapter, available information
is presented on the range of human exposure and intake for endothall from
drinking water, food, and ambient air for the 70-kg adult male. It is not
possible to provide an estimate of the number of individuals experiencing
specific combined exposures from those three sources.
A. EXPOSURE ESTIMATION
1. Drinking Water
No data were obtained on levels of endothall in drinking water. However,
an interim tolerance of 200 pg/L was established for residues of endothall in
IV-1
-------
potable water from the use of Its potassium, sodium, d1-N,N-dimethylalkyla-
mlne, and mono-N,N-dimethyla1ky1amine salts to control aquatic plants In
canals, lakes, ponds, and other potential sources of potable water (U.S. EPA,
1986). The maximum Intake of endpthall from drinking water following such use
was estimated using the tolerance. Assuming that a 70-kg adult male consumes
2 liters of water/day, a maximum Intake of 5.7 ?g/kg/day was calculated.
However, It should be noted that the Intake of endothall 1n drinking water
following other uses may be higher than the maximum calculated here. In
addition, the value presented does not account for variances In Individual
exposures or uncertainties In the assumptions used to estimate exposure.
2. Diet '
No data were obtained on the dietary Intake of endothall In the United
States. Tolerances set by EPA for endothall In and on raw agricultural
commodities are as follows (U.S. EPA, 1986a):
Commodity,,.
cottonseed
potatoes
rice, grain
rice, straw
Tolerance f
100
100
50 (negligible residues)
50 (negligible residues)
These data cannot be used, however, to estimate typical dietary intake.
3.
No data were obtained on levels of endothall in ambient air. Therefore,
the intake of endothall from ambient air could not be estimated.
B. SUMMARY
Data on the intake of endothall from drinking water, food, and ambient
air are insufficient for use in determining which of these three sources is
the major contributor to total intake-.
IV-2
-------
V. HEALTH EFFECTS IN ANIMALS
A. SHORT-TERM EXPOSURE
1. Lethality
Estimates of the acute oral LDSO values for endothall (technical) and
endothall formulations 1n rats range from 31 to 138 mg/kg. The data are
summarized in Table V-l. The range of acute oral LDSO values in rats may have
resulted from different absorption rates for the various endothall salts
tested or may be attributable, to unspecified structural isomers that might
have been present as contaminants in the various formulations employed.
2. Other Effects
a. Oral studies
Brieger (1953c) reported a study in which groups consisting of 20 male
and 20 female rats were fed either 1,000 or 10,000 mg disodium endothall/kg in
the diet (approximately 40 or 400 mg endothall ion/kg/day, assuming.a body
weight of 0.40 kg and daily food consumption of 20 g). Slight degeneration of
liver parenchyma and focal hemorrhagic areas in the kidneys were reported for
male and female rats dosed orally with approximately 40 mg endothall
ion/kg/day for 4 weeks; most of the rats receiving approximately 400 mg
endothall ion/kg/day died within one week.
b. Intravenous studies
Srensek and Woodard (1951) reported (in an abstract) the effects of
intravenous injection of endothall in several species. Dogs that were
administered an iv injection of 5 to 10 mg endothall/kg bw responded with
V-l
-------
Table V-l. Summary of Oral Lethality Data on Endothall In Rats
X Acid
Form tested equivalent Sex
Eridothall 75-86 — »
(technical)
Endothall — M
(technical)
Endothall — -' F -
(technical)
Aquatol K* 28.6 H
(di potassium
endothall)
Aquatol* 15.5
(di sodium
endothail)
Hydrotho! 191* 23.4
{monococoamine
endothall)
Hydout* (mono- 10.0
cocoaml ne
enthothall)
U>J8
ing" formulation/ mg endothall
kg bw Ion/kg bw
51 38-44
57
46 — "
125 36
198-329 31-51
560-590 131-138
GOO 60
Reference
U.S. EPA (1981a)
Gains and
Under (1986)
Gains and
Under (1986)
U.S. EPA (198U);
U.S. EM (19Sla)
U.S. EPA (198Ia)
U.S. EPA (1981a)
'Not specified.
V-2
-------
immediate scratching of the nose, intermittent retching, and vomiting. Some
animals recovered, and some died of respiratory failure. Electrocardiograms
demonstrated no striking changes until respiration ceased. Cats reacted
similarly (dose not reported). Domestic fowl were insensitive to doses four
times greater than those producing toxic effects in dogs and cats (no data
presented}. Rabbits given 25 to 50 mg/kg iv pawed at their noses, developed
irregular breathing and, after 80 to 130 minutes, died of respiratory failure.
Goldstein (1952) reported in an abstract that intravenous injections of 5
mg endothall/kg were often fatal in both dogs and rabbits, and doses of
10 mg/kg were always fatal in both species. This report suggested the heart
as the primary organ of failure, as evidenced by symptomatology, pathology,
electrocardiogram, blood pressure recording, and respiration rate (data not
presented). These findings are in contrast with those of Srensek and Woodard
(1951), who attributed death of the animals to respiratory failure.
c. Inhalation studies
Since endothall is often applied by spray techniques, inhalation exposure
is a potential hazard. Acute inhalation exposure to endothall formulations at
levels above 20,000 mg/m3 (>3,000 mg endothall ion/m1} resulted in lung
congestion and hemorrhage in exposed rats. A number of deaths resulted from a
high-level exposure to these formulations of 1 to 6 hours' duration
(Table V-2). Exposure to airborne endothall at lower levels produced
respiratory'and eye irritation.
d. Skin and eve irritation
Goldstein (1952) reported that a 1% solution of endothall applied to the
unbroken skin of rabbits produced no effects. The same solution applied to
scarified skin resulted in mild skin lesions. Solutions (10-20%) or applica-
tions of the pure, powdered material to intact or scarified skin resulted in
more severe skin effects, including necrosis, and the death of some treated
animals.
V-3
-------
Table V-2* Acute Inhalation Toxicity of Endothall and Formulations
Aqueous Aerosols)
i
% Acid
Form tested equivalent
Endothall 75-86
(technical)
Aquathol* 15.5
(di sodium
endothal 1 )
Aquathol* 15.5
(di sodium
endothal 1)
Aquathol* 28.6
(dipotassium
endothal 1)
Exodsure (mct/nrM
Endothall
Formulation ion Species
50 40 Guinea
- pig
200 31 Rat
20,970 3,250 Rat
20,780 5,940 Rat
Effect
(duration of
exposure)
Respiratory arti
eye irritation!;
recovery (6-hoiiir
exposure)
Irritation; il:l
survived (duraij
tion of exposure
not specified) :
Lung congestion,
hemorrhage; 2/ijO
died (1-hour
exposure)
§'
a«ab.u.. »*..>
rats; 4/10 died'
(1-hour exposure)
Hydrothol 191*
(monococoami ne
endothal1)
23.36
22,120
5,170
Rat
Hemorrhage, con-
gestion in most
rats; 4/10 died'
(1-hour exposure)
SOURCE: Adapted from U.S. EPA (1981b).
V-4
-------
Additional studies of endothall and various formulations (Table V-3) also
demonstrated the skin-irritant capacity of this compound. Solutions contain-
ing the sodium or potassium salts were-mildly irritating to rabbits, while
several organic amine salts produced severe skin irritation and, in some in-
stances, fatalities.
Endothall salts produced severe eye irritation in rabbits when small
quantities were applied to the conjunctiva. These data are shown in
Table V-4. All formulations tested were capable of producing corneal damage.
8. LONG-TERM EXPOSURE
Brieger (19535) orally treated nine male dogs (one dog/dose level) with
doses of 1, 2, 3, 5, 10, 20, 30, 40, and 50 mg disodium endothall/kg bw/day
(0.8 to 40 mg endothall ion/kg/day}, in capsules, for up to 6 weeks. All dogs
administered doses greater than 20 mg disodium endothall/kg bw/day died within
3 to 11 days. The remaining dogs were sacrificed at the end of 6 weeks.
Pathological changes were found- in the gastrointestinal tract of all dogs.
These changes, particularly conspicuous in the dogs at the four highest dose
levels, included necrotic areas at the highest dose and varying degrees of
ulceration and edema at the lower doses, and they may have resulted from
contact of the undiluted compound (administered in capsules) with the walls of
the gastrointestinal tract. Other than the gastrointestinal effects, no other
treatment-related findings were observed.
Brieger (1953a) reported the results of a 2-year feeding study in Wistar
rats. Groups of 10 male and 10 female rats were fed diets containing 0, 100,
300, 1,000, or 2,500 mg disodium endothall/kg of diet (approximately. 0, 4, 12,
40, or 100 mg endothall ion/kg bw/day, respectively, assuming food intake of
20 g/day and mean body weight of 0.4 kg). No toxic effects, other than
lethality, were reported, but the use of a larger number of animals in each
group could have given a stronger assessment. Mortality by the end of the
study was 60 to 90% for all groups except for the high-dose female group,
where mortality was only 30%. The initial group of female controls was
V-5
-------
Table V-3. Primary Dermal Irritation of Endothall and Formulations in
% Acid
Form tested equivalent
Endothall 1-10
(Di sodium)
Aquathol* 15.5
Aquatho! K® 28.6
Hydrothol 191* 23.4
Hydout® 10.0
Dose
Endothall
Formulation ion (mg) Effect
0.5 ml 5-50 Irritation; no
sensitization
0.5 ml 77.5 . No irritation at 24-72
hours
0.5 ml 143 Slight erythema and
edema in 1/6 rabbits at
24 hours; negative at
72 hours
0.5 mL 117 6/6 animals died at 24
hours
0.5 g 50 2/6 abraded skin sites;
severe irritation
SOURCE: Adapted from U.S. EPA (1981a).
V-6
-------
Table V-4. Primary Eye Irritation of Endothall and Formulations in Rabbits
Form tested
Dose
% Acid Endothall
equivalent Formulation ion (mg)
Effect
Endothall
(Technical)
Aquathol®
(Disodium
endothall)
Aquathol K®
(Dipotassium
endothall)
75-86
0.1 g
15.5
0.1 Hi
28.6
0.1 ml
Hydrothol 191® 23.36
(Monococoamine
endothall)
0.1 ml
75-86 In unwashed eyes:
conjunctiva! opacity;
lethality within 24
hours. In washed eyes:
reversible conjunctival
inflammation or a
prolonged reaction with
corneal opacification.
15.5 Severe conjunctival
inflammation, chemosis;
delayed corneal
clouding. No
differences between
washed and unwashed
eyes.
28.6 Conjunctival
irritation; irre-
versible corneal
clouding at 7 days in
unwashed eyes.
Reversible conjunctival
inflammation and iridal
congestion in washed
eyes.
23.4 Corrosive; 3/6 dead at
72 hours.
SOURCE: Adapted from U.S. EPA (1981b),
V-7
-------
reduced to eight rats because one escaped and another accidentally died.
However, the surviving animals were maintained until they were 27 months old.
Brieger considered age of death for each rat, and ranges of mean (t standard
deviation) ages of death were 20.1 to 23.8 (t 2.8 to 6.1) months for all male
groups and 18.9 to 21.6 (t 5.6 to 7.0) months for all female groups. Due to
the high mortality and the small number of animals, this study is considered
to be Inadequate for assessment of endothall toxicity.
Keller (1965) reported the results of a 24-month.study on the effects of
d1sodium endothall in male and female beagle dogs. Purebred beagles (6 to
9 months of age) were divided into four groups of six dogs each (three males,
three females). One group served as control and received only the basal lab-
oratory diet. The other three groups were fed diets containing 100, 300, or
800 mg disodium endothall/kg diet, respectively. According to the author,
these levels of endothall in the diet correspond to dose levels of approxi-
mately 2, 6, or 16 mg endothall ion/kg bw/day, respectively. In the high-dose
group, the dietary intake was increased from 800 to 1,000 mg disodium endo-
thall/kg diet (20 mg endothall ion/kg bw/day) at the end of the 19th month; to
1,300 mg disodium endothall/kg of diet (26 mg endothall ion/kg bw/day) at the
end of the 20th month; to 1,600 mg disodium endothall/kg diet (32 mg endothall
ion/kg bw/day) at the end of the 21st month; and, finally, to 2,000 mg
disodium endothall/kg diet (40 mg endothall ion/kg bw/day) at the 22nd month.
The study was terminated after 24 months.
No gross signs of toxicity were observed in any test animals throughout
the study. Dealth of one female dog at the intermediate dose level was
attributed to pneumonia. Body weight gains and food consumption were within
normal limits throughout the study. One high-dose dog showed a net weight
loss of about 1 kg. Hematology, liver function, and urinalysis results of t-
est animals were comparable to those of controls throughout the study.
Necropsies at 24 months showed no gross pathology that could be attributed to
the test compound. Organ weights and organ-to-body weight ratios of test
animals in the low-dose group were comparable to those of controls.
Intermediate- and high-dose level dogs exhibited increased eight and organ-to-
body weight ratios of the stomach and small intestine. The effect appeared to
V-8
-------
be dose related.. Microscopic examination of tissue sections revealed no
significant or consistent pathological changes that could be attributed to
ingestion of the disodium endothall in the diet. Changes, such as bile duct
proliferation in the test dogs, were considered to be incidental; they were
comparable to controls and within normal limits. This study, therefore,
identifies a LOAEL of 6 mg endothall ion/kg bw/day and a NOAEL of 2 mg
endothall ion/kg bw/day.
In a more recent 12-month dietary study in dogs, disodium endothall was
fe'd to groups of four male and four female beagle dogs at levels of 0, 150,
450, or 1350 ppm (Greenough et al. 1987). After 6 weeks of dosing, the
dietary level at the highest dose was reduced to 1000 ppm because'of anorexia,
decreased food consumption and body weight loss. Compound intake in the low-,
mid- and high-dose groups was approximately 6, 18 and 35.8 mg/kg/day. Five
high-dose dogs (two males and three females) were sacrificed moribund at 62
and 103 days (males and at 104, 193, and 263 days (females). The sacrificed
dogs had subdued behavior and were in poor condition. Histologically, they
showed acute necrosis of the esophageal epithelium and necrosis of the fundus
and pylorus of the stomach.
After the highest dose had been reduced to 1000 ppm, a partial recovery
of the weight loss was observed, but the overall weight gain remained lower
than in controls. No effects on weight gain were observed at 150 or 450 ppm.
Clinical laboratory studies showed a marked reduction of hemoglobin
concentration in high-dose dogs; this was considered secondary to the
debilitated state of the dogs. Several dogs in the high-dose group also had
elevated levels of serum aspartic aminotransferase and alanine
aminotransferase. At gross examination, the livers of high-dose males and
females were noted with a nodular granular surface, and ascites were apparent
in the abdominal cavity. Oval cell hyperplasia in the portal tract and
hepatocyte shrinkage was observed histologically in the liver of all high-dose
dogs and 6/8 dogs fed 450 ppm endothall. No effects on the liver were
observed at the lowest dose tested. No necrotic changes in the esophagus or
stomach were observed in high-dose animals that survived, but reactive
hyperplastic changes were observed in the gastric mucosa of the stomach.
V-9
-------
Similar but less severe histologic findings were observed in all eight dogs
fed 450 ppm. At 150 ppm, very mild injury to the epithelium of the stomach
with Separative hyperplasia" was observed.
Based on the histologic changes in the liver and reactive hyperplastic
response in the gastric mucosa, the LOAEL .is 450 ppm (14.4 mg/kg endothall
ion/day), and considering the marginal effects on the stomach at the lowest
level, the NOAEL is probably slightly lower than 4.8 mg/kg endothall ion/day.
C. REPROOUCTIVE/TERATOGENIC EFFECTS
A three-generation study on the effects of disodium endothall on
reproduction in rats was reported by Scientific Associates (1965). The study
utilized 120 Sprague-Dawley-derived weanling rats (40 to 55 g). Groups of
20 females and 10 males were fed 0, 100, 300, or 2,500 ppm disodium endothall
(approximately 0, 4, 12, or 100 mg endothall ion/kg/day, respectively). Diets
were available a4 libitum throughout the study. Animals were housed indi-
vidually, except during mating. When the animals were 100 days old, they were
bred to produce F1t litters. Ten days after weaning the Ft. progeny, the
animals were rebred to produce F,B litters; 20 female and 10 male F,b weanlings
were selected to continue the study. Using similar procedures, F,b progeny
were bred to produce Fa and F2b litters, and F2i) progeny were bred to produce
Fj. and FJ6 litters. Selected F3b weanlings were necropsied and examined histo-
logically. Observations for all litters included litter size, pup weight, pup
survival, 'sex distribution, and gross abnormalities.
Parental toxicity In this study was demonstrated at the high-dose level
by unthriftiness, reduced food "acceptance" (the animals pawed through their
food), reduced body weight gains, olive to dark-brown discoloration of the
kidneys, and pale adrenals. Reproductive or developmental toxicity was
demonstrated at the high-dose level by reduced litter size, pup weight, and
pup survival, and toxic effects in offspring included emaciation, tremors,
nasal scabs, diarrhea, or peri anal necrosis. Excessive pup mortality at the
high-dose level resulted in only one surviving Frt offspring; this dose level
was .therefore terminated. At the mid-dose level,, no parental toxicity was
L
v-io
-------
observed. However, at the raid-dose level, signs of reproductive/developmental
toxlclty Included reduced pup survival in the F18, f^, and FJb litters; "poorer
quality" of survivors was noted at weaning. No adverse effects were attri-
buted to endothall at the low-dose level. This study provides a NOAEL and a
LOAEL of 12 and 100 mg endothall ixin/kg/day, respectively, for parental
toxicity, and a NOAEL and a LOAEL of 4 and 12 mg endothall ion/kg/day,
respectively, for developmental/reproductive toxicity.
In a teratogenicity study, 4 groups of 40 female Sprague-Oawley-derived
rats (202 to 273 g) were mated and then fed, by gavage, 0, 10, 20, or 30
mg/kg/day aqueous endothall (technical 89.5% acid equivalent) on days 6 to 19
of gestation (Science Applications, Inc., 1982). Doses of test material were
prepared by using a base factor of 1.12 to adjust for purity. On day 20, most
females (25 or 26 per group) were killed, and their litters were delivered by
cesarean section. Implantation data were recorded, and fetuses were weighed,
sexed, and examined for alterations. The remaining dams were allowed to
deliver, and 10 to 13 litters per group were examined for postnatal
development and behavior. Examinations included physical appearance and
development, body weight, eye opening, surface righting reflex (pups were
placed on their backs and allowed 15 seconds to right themselves), and
pivoting locomotion (change in compass direction of the body midline produced
by forelimb motion). The pups were necropsied on postnatal day 21. Two dams
died at the 20-mg/kg bw/day dose, and 10 dams died at the 30-mg/kg bw/day dose
level. No clinical signs were noted prior to death, and no gross lesions were
observed at necropsy. Endothall (technical) was not embryotoxic or terato-
genic at maternal doses of 30 mg/kg bw/day or below, and no patterns of
behavioral dysfunction were observed in any group. The researchers concluded
that the lack of developmental toxicity at dose levels that were fatal to a
number of dams indicated that the maternal organism is more susceptible to
endothall than the conceptus. This study provides a NOAEL of 30 mg endothall
technical/kg bw/day for developmental toxicity, and a NOAEL and a LOAEL of 10
and 20 mg endothall technical/kg bw/day, respectively, for maternal toxicity.
V-ll
-------
In another study, endothall technical (89.5% acid equivalent) was
administered by gavage to 4 groups of 25 pregnant Charles River CD-I mice on
gestation days 6 though 16 at doses of.O (control), 5, 20, and 40 mg/kg bw/day
(IRDC, 1981). Doses of test material were prepared using a base factor of
1.12 to adjust for purity. Blended whole egg with water (4:1, v/v) was used
as the vehicle. Dams were killed on day 17, and the numbers of uterine
implantations were recorded. Fetuses were weighed, sexed, and examined for
alterations. Maternal mortality occurred in 0/25, 0/25, 2/25, and 8/25
females in the control and low-, mid-, and high-dose groups, respectively. In
surviving dams, no signs of maternal toxicity were reported. The incidence of
vertebral and rib malformations in the progeny was increased, but was not
statistically significant, at 40 mg/kg bw/day. Although the effect was not
statistically significant, the authors suggest the results of this study
indicate that endothall was teratogenic at 40 mg/kg bw/day because the
incidence of vertebral and rib malformations in their laboratory is extremely
low. They also concluded that since the malformations were produced at a dose
level that was lethal to approximately 30% of the pregnant females, the
possibility of other factors (maternal toxicity) involved in the etiology of
the reported malformations could not be ruled out. No other signs of
developmental toxicity were demonstrated at the lower doses. This study
provides a NOAEL and a LOAEL for endothall technical for maternal toxicity of
5 and 20 mg/kg bw/day, respectively, and a NOAEL and a LOAEL for developmental
toxicity of 20 and 40 mg/kg bw/day, respectively.
D. MUTA&ENICITY
Relatively few studies investigating the genotoxic potential of endothall
have appeared in the published literature. This section includes the
published experiments as well as a series of unpublished assays that were
performed to meet U.S. EPA registration requirements. These are categorized
into gene mutation assays (Category 1), chromosome aberration assays (Category
2), and studies that assess other mutagenic mechanisms (Category 3). The
findings are discussed below and summarized in Table V-5.
V-12
-------
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1. Gene MutationAssays (Category 1)
a. Reverse mutation in orokarvotes
Andersen et al. (1972) surveyed 110 herbicides including the commercial
grade of endothall in spot tests with eight unidentified histidine-requiring
mutant strains of Salmonella tvphlmurium. Endothall (dose not reported) was
listed as negative; however, only qualitative results.were presented.
Schechtman et al. (1980a) performed an S. tvohimurium mammalian microsome
reverse mutation assay (Ames test) on crystalline endothall (89.5%). Results
of the plate incorporation assay indicated that five doses (50 to 5,000
Mg/plate), either with or without Aroclor 1254-induced rat liver S9, were
neither cytotoxic nor mutagenic in $. tvphimurium TA1535, TA1537, TA1538,
TA98, or TA100.
b. Lower eukarvotes
The potential of endothall (purity not given) to induce recessive lethal
mutations in Neurosoora crassa was evaluated by Sandier and Hamilton-Byrd
(1981). No mutations were observed following a 5-day exposure of asexual
spores, either in suspension or on solid medium, to five doses ranging from
200 to 12,800 ppm. The slower than normal rate of cell growth in the
12,700-ppm.suspension and 12,800-ppm solid medium treatment groups suggests
that cytotoxicity was achieved at these levels.
c. Mammalian cells
A crystalline preparation of endothall (89.5%) was investigated by
Schectman et al. (1980b,c) for the potential to Induce ouabain-resistant
(ouan) mutants in the in vitro BALB/3T3 mouse embryo cell forward mutation
assay. Based on a preliminary cytotoxicity screen, cells in suspension
cultures were exposed to 1.0, 3.0, or 10.0 *g/mL 1n the absence of S9 activa-
tion or 0.01, 0.1, and 1.0 /ig/mL in the presence of S9 activation. The S9
V-16
-------
activation system was obtained from liver of Arochlor 1254-induced rats.
Following treatment, cells were plated directly for cytotoxic effects or
allowed a recovery period and subsequently selected for oua" mutants. In both
the presence and absence of S9 activation, endothall was assayed to a cyto-
toxic dose with-no increase in oua" colonies compared to controls.
d. Sex-linked recessive lethal mutations in Drosophila me!anooaster
Sandier and Hami Iton-Byrd (1981) performed sex-linked recessive lethal
mutation studies with endothall (purity not given). The larval feeding
portion of the study was conducted with 50, 100, and 200 ppm, and the adult
feeding was performed with 400, 800, and 1,600 ppm. Dose selection for both
assays was governed by preliminary results showing reduced survival for both
growth stages at high levels.
Following the larval and adult feeding exposures, males were collected
and mated with untreated females. F, progeny were permitted one round of
brother-sister mating, and the resulting progeny (FJ were scored for lethal
mutations. In the larval feeding study, which represents germ cells treated
in the early stages of spermatogenesis, slight but neither dose-related nor
significant increases in the lethal frequencies were observed at 50 and
100 ppm. Based on the control frequency and sample size (approximately
2,600/group), the slight increases should not be considered to indicate a
positive response. No significantly increased lethal frequencies were
observed in the adult feeding study. Progeny resulting from this exposure
correspond to germ cells that were primarily sperm at the time of treatment.
The combined results of these studies indicated that endothall treatment of
germ cells at different stages of spermatogenesis did not elicit a mutagenic
response.
In another sex-linked recessive lethal assay, Wilson et al. (1956)
exposed adult male D. melanooaster to-an unreported vapor concentration of
19.2% endothall for 24 hours. Following treatment, males were serially mated
with untreated females for 9 days; three brother-sister paired matings were
V-17
-------
allowed for the F, progeny, and the F, progeny were scored for lethal
mutations. The same authors also conducted larval feeding studies with 100
and 250 ppm endothall (19.2%). Eggs were collected, and surviving males were
mated with untreated females as described. From the results, Wilson et al.
concluded that "even by conservative estimates, endothall must be considered
as a mutagen when administered either as a vapor or a food ingredient."
However, the authors based this conclusion on findings from extremely small
sample populations. For example, the highest percentage of induced sex-linked
recessive lethals (0.8%) occurred in the vapor exposure study with adult
males; this value was derived from one mutation in 128 flies (chromosomes)
tested. When the test results were compared to the control (0 mutations in
64 chromosomes), the increase was reported to be approximately ninefold.
Interpretation of JJ. melanooaster sex-linked recessive lethal assay results
relies heavily on sample size, since the precision of the assay to provide
meaningful information is directly related to the background frequency of
spontaneous mutants and the number of tests (chromosomes examined} performed.
Wurgler et al. (1977) developed sample size tables to determine the power of
the test and the degree of confidence in the result. Comparing the data of
Wilson et al. (1956) against these tables shows that at least 1,000 chromo-
somes in both treatment and control groups should have been analyzed for the
endothall-vapor study results to have any biological significance. There is a
similar problem of inadequate sample size with the larval feeding study data.
The findings of this study should be considered inconclusive evidence of a
mutagenic response.
2. Chromosome Aberration Assays (Category 2)
a. Somatic cells fin vivo)
Dietary preparations containing 150, 300, and 600 ppm disodium endothall
(15.8% acid equivalent) were administered to 12-week-old male rats of strain
CRL:COBS CD(SO) Br (five/group) for 5 days in the bone marrow cytogenetic
assay performed by Brusick (1977a). 'Animals were sacrificed 6 hours after
final dosing, bone marrow cells were harvested, and prepared metaphases
V-18
-------
(40 to SO/animal) were scored for structural chromosome aberrations. The
administered doses were neither toxic, cytotoxic, nor clastogenic. The lack
of toxicity at the highest dose, however, indicated that a maximum tolerated
dose (MTD), which could have provided a stronger test, was not achieved.
b. Germinal cells (in vivoh dominant lethal assay
Brusick (1977b) also used similar doses of disodium endothall in conjunc-
tion with a 5-day feeding regime to determine potential clastogenic effects on
germinal cells of male rats of strain CRLrCOBS CD(SO) Br. Following exposure,
males were mated sequentially with untreated females for 7 weeks.- The number
of females in treatment groups ranged from 10 to 18. No relevant increase in
dominant lethal parameters was observed from matings timed to correspond with
the entire period of spermatogenesis; however, the test doses were not overtly
toxic. Similarly, no adverse effects on fertility or total implantation rates
were seen. However, this study is weakened by the low number of matings
(20/group/week) and the inability to establish that an MTD was achieved or
that the test material reached the target cell (gonads).
V
3. Other Mutaoenic Mechanisms (Category 3)
a. Aneuoloidv
Roots of AIlium ceoa. Vicia faba. and Pi sum sativum var alaska were
exposed to'o.l, 0.2, 0.5, and 1.0 ppm endothall (19.2%) (Wilson et al.', 1956).
Results were presented only for P. sativum. but the authors stated that the
findings for the other plants were comparable with those for £. sativum.
Although the high dose suppressed mitosis, suggesting cytotoxicity, no defini-
tive "C-mitotic" effect, which is an indirect measurement of aneuploidy, was
observed.
V-19
_
-------
b. |n vitro sisterchromatid exchange
Vigfusson (1981) exposed human lymphocytes to four concentrations
(0.01 to 10 *g/mL) Aquathol K* (potassium endothall, 28.6% endothall Ion),
both 1n the presence and absence of S9 activation system obtained from rat
liver (it was not specified whether or not the rats had been pretreated with a
microsomal enzyme inducer). Higher doses were completely cytotoxic; at
10 tig/ml (+/- S9), a slight cytotoxlc effect was reported. Following treat-
ment, cells were permitted one round of replication, harvested, stained, and
evaluated for sister chromatid exchange (SCE) Induction (50 metaphase cells/-
dose). Slight but not significant elevations in SCEs were scored-at nonacti-
vated 1.0 ng/mL and 59-activated 10 #g/mL. The remaining doses were negative.
c. In vitro transformation
Doses of Aquathol K® (28.6% endothall Ion) ranging from 1.25 to 50 nL/mL
(approximately 0.36 to 14.3 ng/mL endothall) without exogenous metabolic
activation (Table V-6) and concentrations of endothall spanning a range of
0.78 to 25 nL/mL (approximately 0.22 to 7.2 /»g endothall/mL) in the presence
of freshly isolated rat hepatocytes (Table V-7) were evaluated in the BALB/3T3
In vitro transformation assay by Rundell and Matthews (1981). Results of a
preliminary screening test, to establish dose levels for the transformation
assay, showed that nonactivated doses greater than or equal to 62.5 nL/mL
caused severe reductions in cell survival; levels greater than or equal to
31.25 nL/mL were cytotoxlc. Monolayer cultures either with or without rat
hepatocytes were exposed to the selected doses of Aquathol K?, washed, allowed
a 4-week incubation period, stained, and scored for the number of transformed
foci. In the absence of metabolic activation, endothall produced a signifi-
cant (p <0.01) increase in transformed foci at a single dose (12.5 nL/mL).
V-20
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Table V-6. Effect of Aquathol K on Transformation of BALB/3T3 Cells
Without Metabolic Activation
Test material
Approximate
dose (ng endo-
thall/mL,
Number
of foci
Number of
foci/plate
Number of
pi ates
with foci
Aouathol K« fnL/mll
0
1,
6.
12.5
25.0
50.0'
25
25
0
0.36
79
60
20
14.40
Positive Control
3-Methylcholanthrene
5.0 »g/ml_
7
9
6
19**
5
2
33*
Initial Assay
0.25
0.56
0.38
1.19
0.31
0.13
6/28
9/16
6/16
10/16
5/16
2/16
2.2
15/16
Repeat Assay
Aouathol K® fnL/mU
0
2.0
4.0
8.0
16.0
32.0
Positive Control
3-Methylcholanthrene
5.0
0
0.57
,14
.20
.56
1,
2.
4.
9.12
8
5
15**
16**
2
1
0.21
0.26
0.75
1.80
0.11
0.05
6/39
5/19
7/20
9/20
2/19
1/19
49
**
2.45
20/20
'Higher doses were found to be cytotoxic (<10% survival) in preliminary
studies.
"Significantly different from the negative control at p sO.Ol.
SOURCE: Adapted from Rundell and Matthews (1981).
V-21
-------
Tab"v'7-
Test material
Aouathol K* fnl
0
0.78
3.13
6.25
12.50
25.00-
Approximate
dose (ng endo-
thall/mL)
./ml)
0
0.22
0.90
1.79
3.60
7.20
Number
of foci
39
82**
56**
91**
53**
44**
Number of
foci/plate
1.3
5.13
4.31
6.07
3.31
2.75
Number of
plates
with foci
22/23
16/16
13/13
15/15
14/14
16/16
Positive Controls
Oimethylnitrosamine
1.25 ML/mL
Cyclophosphanride
2.2 *g/mL
103
133
6.44
8.31
16/16
16/16
'Higher doses were found to be cytotoxlc (<10% survival) In preliminary
studies.
**Sign1ficantTy different from the control at p sO.Ol.
V-22
-------
Confirmation of this response was demonstrated at two levels (4 and 8 nL/mL)
in a repeat study (Table V-6); however, the transformation frequency was
comparable at both dose levels. In the presence of rat hepatocytes, signifi-
cant (p <0.01) but not dose-related increases in transformed foci were scored
over the entire 0.78 to 25 nL/mL treatment range; the greatest increase
(6.07 foci/dish) occurred at 6.25 nL/mL. The frequency at the 6.25 nL/mL
level is only slightly lower than that calculated for the positive control,
dimethylnitrosamine (DMN) at 1.25 «L/mL. The authors concluded that
Aquathol K® both with and without rat hepatocyte-medfated activation was
active in the BALB/3T3 in, vitro transformation assay.
Although Aquathol K® was assayed in accordance with generally accepted
procedures, the findings of this study can be questioned for the following
reasons: (1) the effect was not dose related; (2) the response elicited in
the presence of primary rat hepatocytes suggests that Aquathol K* is com-
parable in transforming activity to the potent carcinogen, DMN (the response
at 6.25 nL/mL (1.8 »g endothall ion/mL) is actually of a higher magnitude than
DMN at 1.25 ML/mL (approximately 92.6 *ig/mL)); (3) the in vitro results are
not supported by the findings of long-term carcinogenicity bioassays and
mutagenicity tests with endothall; and (4) the rat hepatocyte metabolic
activation phase for this test system has not been validated (Heidelberger
et al., 1983).
In addition, the possibility that other components in the preparation may
have been responsible for the positive response should not be discounted.
Studies published by Brusick (1986) indicate that excess monovalent ions such
as Na* and K*, which are introduced into the culture medium when salts of test
compounds are assayed, have led to erroneous false positive results; transfor-
mation in BALB/3T3 is responsive, to this ionic effect. Brusick (1986) further
indicated that "false positives" in the in vitro transformation assay could be
attributed to ionic alterations in the treatment conditions. Although the
findings of this study are questionable, 'no definitive conclusions can be
reached; further testing is required to clarify, the potential, if any, of
endothall to induce in vitro cell transformation.
V-23
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E. CARCINOGENICITY
In a 2-year study, Brieger (1953a) exposed 10 male and 10 female rats per
dose level to endothall in the diet at levels ranging from 100 to 2,500 mg
disodium endothall/kg food (about 4 to 100 mg endothall ion/kg/day, assuming
food intake of 20 g/day and mean body weight of 0.40 kg). After 2 years,
there were no differences in the number or types of tumors in exposed animals
in comparison to control animals. Two treated male rats developed lung
tumors, but statistical significance of their presence was not assessed and
tumor type and dose group were not given. As previously indicated in Section
V.B, Long-term Exposure, this study by Brieger (1953a) is deficient owing to
the small number of animals available for assessment.
F. SUMMARY
The acute oral toxicity LDM values for endothall ion in various
endothall formulations range from 31 to 138 mg endothall ion/kg bw. Single
intravenous doses of 5 mg endothall/kg bw or higher were fatal to dogs and
rabbits. Death was attributed to either respiratory or cardiac failure.
Inhalation exposure, at levels above 20,000 mg endothal1/m3 for 1 hour,
resulted in lung congestion and hemorrhage in rats. Endothall is irritating
to the skin and corrosive to the eyes of rabbits.
Rats that received oral doses of approximately 400 mg endothall ion/kg
bw/day died within 1 week. The animals exhibited slight liver degeneration
and focal hemorrhagic areas in the kidneys at necropsy.
Oral administration of disodium endothall to male dogs in doses ranging
from 0.8 to 40 mg endothall ion/kg bw/day for up to 6 weeks produced patho-
logical changes in the gastrointestinal tract of all dogs. These changes
ranged from inflammation to necrosis, and may have resulted from contact of
the undiluted compound (administered in capsules) with the walls of the
gastrointestinal tract.
V-24
-------
In a chronic study, no gross signs of toxicity were evident in dogs dosed
with 2, 6, or 16 mg endothall ion/kg bw/day and necropsied after 24 months.
Weight gain, food consumption, hematology, liver function tests, and urinaly-
ses were comparable in treated and-control groups. The organ weight and
organ-to-body weight ratios of the stomach and small intestine were increased
above those for controls for the intermediate- and high-dose level animals.
Microscopic examination did not indicate changes that could be attributable to
the ingestion of the compound in the diet. The low-dose level of 2 mg endo-
thall ion/kg bw/day was the NOAEL, whereas the 6-mg endothall ion/kg bw/day
dose level produced changes in organ weight and was considered to be the LOAEL
in this study.
A 2-year toxicity feeding study with disodium endothall in rats at an
intake of 0, 4, 12, 40, or 100 mg endothall ion/kg bw/day was available for
evaluation. The study used a small number of animals in control and treated
groups, and lethality was reported at all doses tested. This study was
inadequate to evaluate toxicity or set an effect level.
In a three-generation reproduction study in rats fed endothall at 4, 12,
and 100 mg endothall ion/kg bw/day, no apparent effect was observed in dams or
any offspring at the lowest dose. At approximately 12 mg endothall ion/kg
bw/day in the diet, reduced pup survival and "poor quality" of weanlings were
noted, and at 100 mg endothall ion/kg bw/day in the diet, reduced litter size,
pup weight, and pup survival were noted. This study provides a NOAEL for
reproductive-effects of 4 mg endothall ion/kg bw/day and a LOAEL for reproduc-
tive effects of 12 mg endothall ion/kg bw/day.
Results from a teratology study in rats indicated that endothall
(technical 89.5% acid equivalent) is neither embryotoxic nor teratogenic at
maternal doses of 30 mg/kg bw/day or less, whereas maternal mortality occurred
at 20 and 30 mg/kg bw/day. It appears that the dams are more susceptible to
endothall than the developing conceptus. This study provides for endothall
technical a NOAEL of 30 mg/kg bw/day for developmental toxicity, and a NOAEL
and a LOAEL of 10 and 20 mg/kg bw/day, respectively, for maternal toxicity.
Similarly, in mice, maternal mortality occurred at 20 and 40.mg endothall
V-25
-------
technical/kg bw/day. Malformations were reported at 40 mo/kg bw/day. The
authors stated that more malformations occurred among control animals in their
laboratory; however, they also considered that these effects were associated
with a high incidence of maternal-mortality. The NOAEL and LOAEL for maternal
toxicity are 5 and 20 mg/kg bw/day, respectively, and the NOAEL and LOAEL for
developmental toxicity are 20 and 40 mg/kg bw/day, respectively.
Endothall was not mutagenic in bacterial, fungal, mammalian cell, or
Drosophila mutagenicity tests. In vivo somatic or male germinal-cell cyto-
genetic assays performed with the disodium salt showed no clastogenic effects
within the investigated nontoxic dose ranges.
Endothall did not induce aneuploidy in plants, and Aquathol K®
(dipotassium endothall) did not increase the frequency of SCE in human
lymphocytes. Although Aquathol K® exhibited transforming activity in BALB/3T3
cells both in the presence and absence of primary rat hepatocytes, the results
can be questioned because of Issues regarding procedure and Interpretation.
Although endothall has not been shown to be carcinogenic, the present
data base is inadequate to assess its carcinogenic potential.
V-26
-------
VI. HEALTH EFFECTS IN HUMANS
A. CLINICAL CASE REPORTS
The only information on endothall toxicity in humans is a case report of
the suicide of a young male (54 kg) who ingested an estimated 7 to 8 g of
disodium endothall (approximately 102 to 118 mg endothall ion/kg bw)
(Allender, 1983). Repeated vomiting, which may have resulted in some
aspiration of the compound, occurred after ingestion of the solution, which
apparently removed much of the material from the gastrointestinal (GI) tract.
At autopsy, the stomach contents contained only 4 mg endothall/100 mL stomach
contents. Levels of 1.7 and 1.0 mg endothall/100 g tissue were observed in
the liver and blood, respectively. The autopsy report revealed gross hemor-
rhage of the GI tract and widespread focal hemorrhages and edema in the lungs.
The latter may have resulted from aspiration of the compound during vomiting,
since the compound does have a local irritating capacity.
B. EPIDEMIOLOGICAL STUDIES
No epidemiological reports concerning endothall exposure were found in
the literature.
C. HIGH-RISK POPULATIONS
No data identifying any high-risk population were found in the
literature.
D. SUMMARY
Little information was available on human health effects of endothall.
There are no reports of adverse health effects in individuals who manufacture
or apply this compound. A single suicide report indicates that oral ingestion
by humans produces effects observed in animals. The human autopsy report
indicated gross hemorrhage of the GI tract and widespread focal edema and
hemorrhage, of the lung.
VI-1
-------
VII. MECHANISMS OF TOXICITY
A. ANIMALS
The mechanism of endothall toxicity in animals is not known, Goldstein
(1952) reported that the cause of death following intravenous injection of 5
or 10 mg/kg bw in dogs was cardiac arrest. In an earlier study (Srensek and
Woodard, 1951), death in dogs or rabbits dosed with 5 to 50 mg/kg bw was
attributed to respiratory failure. No other data are available on the
mechanism of action in animal species.
B. PLANTS
Studies of the effects of endothall in plants indicate that it may have
several actions. Mann and Pu (1968) reported that endothall (5 *g/mL} caused
an approximate 40% inhibition of incorporation of malonic acid into the lipid
fraction of hypocotyl segments of Hemp sesbania fSesbania exaltata). Maestri
and Currier (1966) suggested that endothall produced a variety of membrane
effects that resulted in wilting and drying of leaf tissue. The authors also
reported increased respiratory rate in leaves sprayed with endothall.
However, these studies do not provide a clear understanding of the mechanism
of action of this compound in plants.
C. SYNERGISTIC/ANTAGONISTIC EFFECTS
Gzhegotskii and Martynyuk (1966) reported on the toxicity of Murbetol, a
combination herbicide containing 142.4 g endothall and 85.5 g isopropy1-
phenylcarbamate (isopropyl N-phenylcarbamate; propham) per liter. Although
specific data were not presented, 14 times as much Murbetol was required to
produce the same toxic response in albino rats, mice, and guinea pigs as
endothall alone. This was attributed to the "antagonistic interaction" of the
components. The endpoints utilized as measures of the toxic response were not
reported.
VIM
-------
0. SUMMARY
No specific Information Is available on the mechanism of toxicity of
endothall in animals. Intravenous toxicity studies in dogs suggest that the
cause of death is cardiac or respiratory arrest. One study suggests that
isopropylphenylcarbamate may antagonize endothall toxicity, but no data were
provided.
VII-2
-------
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS .
The quantification of toxicologlcal effects of a chemical consists of
separate assessments of noncarcinogenic and carcinogenic effects. Chemicals
that do not produce carcinogenic effects are believed to have a threshold dose
below which no adverse, noncarcinogenic health effects occur, while carcino-
gens are assumed to act without a threshold.
A. PROCEDURES FOR QUANTIFICATION OF TOXICOLOGICAL EFFECTS
1. Noncarcinoqenic Effects
In the quantification of noncarclnogenic effects, a Reference Dose (RfD),
formerly called the Acceptable Dally Intake (ADI), Is calculated. The RfD Is
an estimate (with an uncertainty spanning perhaps an order of magnitude) of a
dally exposure of the human population (Including sensitive subgroups) that Is
likely to be without an appreciable risk of deleterious health effects during
a lifetime. The RfD Is derived from a No-Observed-Adverse-Effect Level
(NOAEL), or Lowest-Observed-Adverse-Effect Level (LOAEL), Identified from a
subchronic or chronic study, and divided by an uncertainty factor(s). The RfD
is calculated as follows:
Rfd » fNOAEL or LQAEL1
Uncertainty factor(s)
mg/kg bw/day
Selection of the uncertainty factor to be employed in the calculation of
the RfD is based on professional judgment while considering the entire data
base of toxicological effects for the chemical. To ensure that uncertainty
factors are selected and applied in a consistent manner, the Office of
Drinking Water (ODW) employs a modification to the guidelines proposed by the
National Academy of Sciences (NAS, 1977, 1980) as follows:
An uncertainty factor of 10. is generally used when good chronic or
subchronic human exposure data identifying a NOAEL are available and
VIII-1
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are supported by good chronic or subchronic toxicity data in other
species.
• An uncertainty factor of 100 Is generally used when good chronic
toxicity data identifying a NOAEL are available for one or more
animal species (and human data are not available), or when good
chronic or subchronic toxicity data identifying a LOAEL in humans
are available.
* An uncertainty factor of 1,000 is generally used when limited or
incomplete chronic or subchronic toxicity data are available, or
when good chronic or subchronic toxicity data identifying a LOAEL,
but not a NOAEL, for one or more animal species are available.
The uncertainty factor used for a specific risk assessment is based prin-
cipally on scientific judgment rather than scientific fact and accounts for
possible intra- and interspedes differences. Additional considerations,
which may necessitate the use of an additional uncertainty factor of 1 to 10,
not incorporated in the NAS/ODW guidelines for selection of an uncertainty
factor include the use of a less-than-lifetime study for deriving an RfD, the
significance of the adverse health effect, pharmacokinetics factors, and the
counterbalancing of beneficial effects.
From the RfO, a Drinking Water Equivalent Level (OWEL) can be calculated.
The DWEL represents a medium-specific (i.e., drinking water) lifetime expo-
sure, at which adverse, noncarcinogenic health effects are not anticipated to
occur. The DWEL assumes 100% exposure from drinking water. The DWEL provides
the noncarcinogenic health effects basis for establishing a drinking water
standard.
For ingestion data, the DWEL is derived as follows:
DWEL => RfD x fbodv weight in kol - mg/L (
Drinking water volume in L/day
VIII-2
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where:
Body weight -assumed to be 70 kg for an adult.
Drinking water volume - assumed to be 2 L per day for an adult.
In addition to the RfO and the DUEL, Health Advisories (HAs) for
exposures of shorter duration (One-day, Ten-day, and Longer-term) are
determined. The HA values are used as informal guidance to municipalities and
other organizations when emergency spills or contamination situations occur.
The HAs are calculated using an equation similar to the RfO and DWEL; however,
the NOAELs or LOAELs are identified from acute or subchronic studies. The HAs
are derived as follows:
HA
fNOAEL or LQAEL1 x fbwl
X (.
L/day)
Using the above equation, the following drinking water HAs are developed
for noncarcinogenic effects:
1. One-day HA for a 10-kg child ingesting 1 L water per day.
2. Ten-day HA for a 10-kg child ingesting 1 L water per day.
3. Longer-term HA for a 10-kg child ingesting 1 L water per day.
4. Longer-term HA for a 70-kg adult ingesting 2 L water per day.
The One-day HA calculated for a 10-kg child assumes a single acute expo-
sure to the chemical and is generally derived from a study of less than 7 days
duration. The Ten-day HA assumes a limited exposure period of 1 to 2 weeks
and is generally derived from a study of less than 30 days duration. The
Longer-term HA is derived for both a 10-kg child and a 70-kg adult and assumes
an exposure period of approximately 7 years (or 10% of an individual's
lifetime). The Longer-term HA is generally derived from a study of subchronic
duration (exposure for 10% of an animal's lifetime).
VIII-3
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2- Carcinogenic Effects "
The EPA categorizes the carcinogenic potential of a chemical, based on
the overall weight of evidence, according to the following scheme:
• Group A: Human Carcinogen. Sufficient evidence exists from epi-
demiology studies to support a causal association between
exposure to the chemical and human cancer.
• Group B: Probable Human Carcinogen. Sufficient evidence of car-
cinogenicity in animals with limited (Group Bl) or inade-
quate (Group 82) evidence in humans.
• Group C: Possible Human Carcinogen. Limited evidence of carcino-
genicity in animals in the absence of human data.
• Group 0: Not Classified as to Human Carcinoqenicitv. Inadequate
human and animal evidence of careinogenicity or for which
no data are available.
Group E: Evidence of Noncarcinogenicitv for Humans. No evidence of
carcinogenicity in at least two adequate animal tests in
different species or in both adequate epidemiologic and
animal studies.
If toxicological evidence leads to the classification of the contaminant
as a known, probable, or possible human carcinogen, mathematical models are
used to calculate the estimated excess cancer risk associated with the inges-
tion of the contaminant in drinking water. The data used in these estimates
usually come from lifetime exposure studies in animals. To predict the risk
for humans from animal data, animal doses must be converted to equivalent
human doses. This conversion includes correction for noncontinuous exposure,
less-than-lifetime studies, and for differences in size. The factor that
compensates for the size difference is the cube root of the ratio of the
VIII-4
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animal and human body weights. It is assumed that the average adult human
body weight is 70 kg, and that the average water consumption of an adult human
is 2 liters of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure via
ingestion of water. The cancer unit risk is usually derived from a linearized
multistage model with a 95% upper confidence limit providing a low-dose
estimate; that is, the true risk to humans, while not identifiable, is not
likely to exceed the upper-limit estimate and, in fact, may be lower. Excess
cancer risk estimates may also be calculated using other models such as the
one-hit, Weibull, logit, and probit. There is little basis in the current
understanding of the biological mechanisms involved in cancer to suggest that
any one of these models is able to predict risk more accurately than any
others. Because each model is based on differing assumptions, the estimates
that are derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an inherent uncertainty due to the systematic and
random errors in scientific measurement. In most cases, only studies using
experimental animals have been performed. Thus, there is uncertainty when the
data are extrapolated to humans. When developing cancer risk rate levels,
several other areas of uncertainty exist, such as the incomplete knowledge
concerning, the health effects of contaminants, in drinking water; the impact of
the experimental animal's age, sex, and species; the nature of the target
organ system(s) examined; and the actual rate of exposure of the internal
targets in experimental animals or humans. Dose-response data usually are
available only for high levels of exposure, not for the lower levels of
exposure closer to where a standard may be set. When there is exposure to
more than one contaminant, additional uncertainty results from a lack of
information about possible synergistic or antagonistic effects.
VIII-5
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B. QUANTIFICATION OF NONCARCINOGENIC EFFECTS FOR ENOOTHALL
1. One-dav Health Advisory
The single oral LDSO values of endothall Ion In various formulations
range from 31 to 138 rag/kg bw (U.S. EPA, 1981a). No studies were found that
Identified a NOAEL or LOAEL based on more sensitive endpoints (i.e., other
than mortality) suitable for derivation of the One-day HA value. In the
absence of adequate data, it is recommended that the Ten-day HA of 800 »g
endothall ion/L be used as a conservative estimate of the One-day HA value.
2. Ten-dav Health Advisory
Table VIII-1 summarizes studies considered for calculation of the Ten-day
HA for endothall. The study by Brieger (1953c) defined a LOAEL of 40 mg
endothall ion/kg bw/day based on gross necropsy and histopathology (slight
liver degeneration and focal hemorrhagic areas in kidneys of rats). However,
the study of Brieger (1953c) defined no NOAEL because no experiments with
doses lower than the LOAEL were performed.
The teratogenicity study in rats (Science Applications, Inc., 1982)
defines NOAELs of 10 and 30 mg endothall technical/kg bw/day for maternal
toxicity and teratogenicity, respectively. The teratogenicity study in mice
(IROC, 1981) defines NOAELs of 5 and 20 mg endothall technical/kg bw/day for
maternal toxicity and teratogenicity, respectively.
The higher NOAELs of 20 and 30 mg endothall/kg bw/day for teratogenicity
in mice and rats, respectively, are not considered in calculating the Ten-day
HA, because they are equal to or greater than the LOAEL of 20 mg endothall/kg
bw/day for maternal toxicity in mice (IRDC, 1981) and in rats (Science Ap-
plications, Inc., 1982).
VIII-6
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The NOAEL of 10 mg endothall technical (89.5% acid equivalent)/kg bw/day
for maternal toxlclty in rats (Science Applications, Inc., 1982) is therefore
selected for calculation of the Ten-day HA. The selected value is higher than
the NOAEL of 5 ing endothall/kg bw/day for maternal toxicity in mice (IRDC,
1981), and is without significant fetotoxicity or teratogenicity. Although
10 mg/kg/day was not tested in mice, the similarity of maternal toxic effects
in the mice and rats at 20 mg/kg/day (deaths of 2/25 rats and 2/25 mice)
supports the use of the higher NOAEL between these studies.
Using a NOAEL of 10 mg endothall/kg bw/day, the Ten-day HA for a 10-kg
child is calculated as follows:
Ten-day HA » flO mQ/ko/davmO ko) • 1.0 mg/L = 0.8 mg endothall
(100)(1 L/day) ion/L (800 ,.g/L)
where:
10 mg/kg/day
10 kg
100
NOAEL,based on absence of maternal or fetal toxicity
in mice exposed to endothall technical (89.5% acid
equivalent), adjusted for purity to 10 mg/kg/day, via
gavage during days 6 to 16 of gestation.
assumed weight of a child.
uncertainty factor, chosen in accordance with NAS/ODW
guidelines for use with a NOAEL from an animal study.
1 L/day = assumed water consumption by a 10-kg child.
3. Longer-term Health Advisory
The DUEL adjusted for a 10 kg child (200 »g/L) is proposed for use as a
Longer-term HA. The proposed Longer-term HA for a 70-kg adult is the DWEL
(700 ng/l). The studies summarized in Table VIII-2 that are considered for
derivation of the DWEL can also be considered for calculation of the Longer-
term HA because the duration of both studies is well below the lifespans of
the species tested. On this basis, the 2-year feeding study in dogs by Keller
(1965) is preferred over the reproduction study in rats by Scientific
VIII-8
-------
Associates (1965) which although each generation was on study for 100 .days as
compared with the longer duration of 2 years for the dogs, has a somewhat
higher NOAEL and LOAEL.
Similarly, the 2-year study In dogs by Keller (1965) is preferred over
the study of Greenough et al. (1987) because of the longer duration and the
slightly lower LOAEL and NOAEL. >
No existing guidelines or standards were located for longer term (sub-
chronic) exposure to endothall.
4. Reference Dose and Drinking Hater Equivalent Leel-
Table VIII-2 summarizes studies considered for derivation of the RfD and
DWEL for endothall. The study by Keller (1965) identified a LOAEL of 6 mg
endothall ion/kg bw/day and a NOAEL of 2 mg endothall ion/kg bw/day based on
increased organ weights and organ-to-body weight ratios for the stomach and
small intestine. Although no significant histopathological changes were
observed in association with the increased organ weights and organ-to-body
weight ratios, the effect appeared to be dose dependent. In addition, in a
1-year dog study by Greenough et al. (1987), dose-related histologic changes
in the stomach and small intestine ranging from marginal hyperplasia
(4.8 mg/kg/day) to reactive hyperplasia and necrosis (28.6 mg/kg/day) were
observed. However, the NOAEL and LOAEL were higher than in the Keller (1965)
study. Thus, the study of Keller (1965) has been selected as the basis for
derivation of the RfD and DWEL. Similarly, the study by Scientific Associates
(1965), a three-generation study (100 days/generation), with a higher NOAEL of
4 mg/kg/day and a shorter duration, is not used to calculate the RfD and DWEL
values.
Using the study of Keller (1965), the DWEL is derived as follows:
Step 1: Determination of the Reference Dose (RfD)
f? mq/kq/day) „ 0 02 mg endothall/kg/day
(100)
VIII-9
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VIII-IO
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where:
2 mg/kg/day - NOAEL for endothal.l ion, based on absence of Increased
organ weight and organ-to-body weight ratios for the
stomach and small Intestine of dogs exposed to d1sodium
endothall via the diet for 2 years.
100 » uncertainty factor, chosen In accordance with
NAS/ODW guidelines for use with a NOAEL from an
animal study.
Step 2: Determination of the Drinking Water Equivalent Level (DUEL)
DWEL . (0.2 mg/kg/day) (70 kg)
z I/day
endothall/L (700 «g/L)
where:
0.02 mg/kg/day - RfD.
70 kg » assumed weight of an adult.
2 L/day » assumed water consumption of a 70-kg adult.
The DWEL assumes 100% Intake from drinking water. The DWEL may be
reduced when relative source contribution is taken into account.
No existing guidelines or exposure standards were found that relate
directly to human exposure. Several residue tolerances have been established
for crops. These range from 0.05 ppm (negligible residues) for rice, grain,
and straw to 0.1 ppm for cottonseed, hops, and potatoes (U.S. EPA, 1986).
An interim tolerance of 200 #g/L has been published for residues of
endothall, used to control aquatic plants, in potable water (U.S. FDA, 1986).
C. QUANTIFICATION OF CARCINOGENIC EFFECTS FOR ENDOTHALL
1. Categorization of Carcinogenic Potential
VIII-11
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•
The International Agency for Research on Cancer (IARC) and the U.S.
Environmental Protection Agency have not evaluated the carcinogenic potential
of endothall (WHO, 1982). Available toxicity data do not show endothall as
carcinogenic. Endothall can be placed in Group D (inadequate evidence in
humans and animals) by the EPA's guidelines for carcinogenic risk assessment
(U.S. EPA, 1986).
2. Quantitative Carcinogenic Risk Estimates
No quantitative assessment of excess cancer risk has been reported.
D. SUMMARY .
Table VII1-3 summarizes HA and DUEL values calculated on the basis of
noncarcinogenic endpoints. No estimations of excess cancer risk were
performed.
VIII-12
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Gzhegotskii MI, Martynyuk VZ. 1966. Toxicology of the combined herbicide
"murbetol." Hyg. Sanit. 31:225-229.
Heidelberger C, Freeman AE, Prenta RJ, Sivak A, Bertram JS, Casto BC, Dunkel
VC, Francis MW, Kakunaga T, Little JB, Schechtman LM. 1983. Cell transforma-
tion by chemical agents - A review and analysis of the literature. A Report
of the U.S. Environmental Protection Agency Gene-tox Program. Mutat. Res.
114:283-385.
IRDC. International Research and Development Corporation. 1981. Teratology
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U.S. Environmental Protection Agency, Washington, DC.
Keller J. 1965. Two-year chronic feeding study of disodium endothall to
beagle dogs. EPA Pesticide Petition 6G0503. EPA Accession No. 090586,
Exhibit 7.
Maestri M, Currier HB. 1966. Toxic effects of endothall. Plant Physio!.,
Proc. Ann. Meeting. Abstract VII.
Mann JD, Pu M. 1968. Inhibition of lipid synthesis by certain herbicides.
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Rundell JO, Matthews EJ. 1981. Evaluation of Aquathol K« in the In vitro
transformation of BALB/3T3 cells with and without metabolic activation assay.
Report to Metro, Seattle, WA; EPA Accession No. 245680.
Sandier L, Hamilton-Byrd EL. 1981. The induction of sex-linked recessive
lethal mutations in Drosoohila melanooaster by Aquathol K«, as measured by the
Muller-5 test. Report to Municipality of Metropolitan Seattle, Seattle, WA.
Schechtman LM, Cumen RD, Parmar AS, Sinsky PM. 1980a. Activity of T1604 in
the Salmonel1 a/miocrosomal assay for bacterial mutagenicity. Report to
Pennwalt Corp., Tacoma, WA; EPA Accession No. 244126.
Schechtman LM, Beard SF, Sinsky PM. 1980b. Activity of T1604 in the ia vitro
mammalian cell point mutation assay in the absence of exogenous metabolic
activation. Report to Pennwalt Corp., Tacoma, WA; EPA Accession No. 244126.
IX-2
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U.S. EPA. 1987. U.S. Environmental Protection Agency. Endothall health
advisory. Washington, DC: U.S. EPA, Office of Drinking Water.
U.S. FDA. 1986. U.S. Food and Drug Administration. Code of Federal
Regulations. 21 CFR 193.180; April 1.
Vigfusson NV. 1981. Evaluation of the mutagenic potential of Aquathol K* by
the induction of sister chromatid exchanges in human lymphocytes in vitro.
Report to Municipality of Metropolitan Seattle, Seattle, WA; EPA Accession No.
245680.
Weil CS, Condra N, Haun C, Striegel JA. 1963. Experimental carcinogenic!ty
and acute toxicity of representative epoxides. Am. Indust. Hyg. Assoc. J.
24:305-325.
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Research on Cancer Monographs, Vols. 1-29. Supplement 4. Geneva: World
Health Organization.
Wilson SM, Daniel A, Wilson GB. 1956. Cytological and genetical effects of
the defoliant endothall. J. Heredity 47:151-155.
Windholz K, Budavari S, Blumetti RF, Otterbein ES. 1983. The Merck Index.
10th Ed. Rahway, NJ: Merck and Co., Inc., p. 516.
Wurgler FE, Sobels FH, Vogel E. 1977. Drosophila as assay system for
detecting genetic change. In: Kilbey BJ, Legator M, Nichols W, Ramel C, eds.
Handbook of Mutagenicity Test Procedures. Amsterdam, New York, Oxford:
Elsevier Scientific Publishing Co., pp. 335-373.
Yeo RR. 1970. Dissipation of endothall and effects on aquatic weeds and
fish. Weed Sci. 18:282-284.
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