V
January 1992
FINAL
DRINKING WATER CRITERIA DOCUMENT
FOR
DIQUAT
Health and Ecological Criteria Division
Office of Science and Technology
Office of Mater
U.S. Environmental Protection Agency
Washington, DC 20460
csl
cn
HEADQUARTERS LIBRARY
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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January 1992
FINAL
DRINKING WATER CRITERIA DOCUMENT
FOR
DIQUAT
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 FIGURES vii
LIST UF TABLES vii
FOREWORD viii
I. SUMMARY 1,1
II. PHYSICAL AND CHEMICAL PROPERTIES tl-L
III. TOXICOKINETICS ................ '111-1
A. Absorption ' III-l
B. Distribution III-3
C. Metabolism III-4
D. Excretion III-5
E. Bioaccumulation and Retention III-6
F. Summary 111-7
IV. HUMAN EXPOSURE IV-i
A. Exposure Estimation IV-2
1. Drinking Water IV-2
2. Diet lv-2
3. Air IV-2
B. Summary IV-*
V. HEALTH EFFECTS IN ANIMALS V-l
A. Short-term Exposure V-l
1. Lethality V-l
2. Other Effects V-5
3. Subacute Toxicity V-l2
B. Lony-term Exposure
1. Subchronic Toxicity
2. Chronic Toxicity V-l4
C. Reproductive/Teratoyenic Effects V-19
D. Mutagenicity '. V-2*
1. Gene Mutation Assays . :ac*',o-y 1} V-2
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TABLE QF CONTENTS (continued)
VI. HEALTH EFFECTS IN HUMANS
VIII,
A. Clinical Case Studies .
B. Epidemiological Studies
C. Hign-Risk Subpopulations
i). Summary
A. Formation of Free Radicals
B. Lipid Peroxidation and Hole of Oxygen
C. Summary
vi-l
VI-l
VI-4
VI-4
VI-6
VII. MECHANISMS UF TQXICITY VIi-l
QUANTIFICATION OF TOXICULOGICAL EFFECTS
A. Procedures for Quantification of Toxicological Effects . . .
1. .Noncarcinogenic Effects
2. Carcinogenic Effects .. ,
Vll-l
VII-2
Vll-b
vin-i
VIII-l
VIII-l
VIII-4
B. Quantification of Noncarcinogenic Effects .for Uiquat
1. One-day Health Advisory .
2. Ten-day Health Advisory .
3. Longer-term Health Advisory
4. deference Dose and Drinking Water Equivalent Level . . .
C. Quantification of Carcinogenic Effects for Diquat
0. Summary ,
VII
VII
VII
VII
VIIi-11
VIII-13
-o
-d
-y
IX. REFERENCES
IX-1
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LIST OF FIGURES
Fiyure No.
.
II-l Reduction of Diquat to tne Free Radical
LIST OF TABLES .
Table No.
II-l Physical and Chemical Properties of Diquat Dibromide . . . 11-2
IV-1 Tolerances for Oiquat I:/-3
V-l Acute Oral Toxicity of Diquat in Several Animal Speci
es . v-2
V-2 Effects of Diquat on the Eye in a 2-Year Feeding
Study in Rats V-17
V-3 Summary of Results From Selected Mutagenicity Studies . . V-2b
VI-1 Summary of Clinical Case Studies of Diquat Poisoning . . . VI-2
VIII-l Summary of Candidate Studies for Derivation of the
One-day Health Advisory for Uiquat VIII-7
VI11-2 Summary of Candidate Studies for Derivation of the DWEl
for Oiquat VIII-12
VI11-3 Summary of Candidate Studies for Derivation of the
Carcinogenic Risk Estimates for Diquat VIII-ls
VIII-4 Summary of Quantification of Toxicoloyical Effects for
Uiquat VI11-15
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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 expos.ure 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 ii
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I. SUMMARY
Diquat is a dipyridylium herbicide that has been used extensively to
control terrestrial and aquatic weeds. It is commercially available as an
aqueous solution of diquat dibromide (2UO g diquat ion/L) under tie trade name
\
Reglone. Although diquat is stable in water, it is photochemically degraded
in sunlignt. It is strongly adsorbed by soil particles (particularly clays)
and is, therefore, relatively immobile in soils.
Gastrointestinal absorption of diquat in rats is low. Following oral
administration of l^C-diquat to rats, most of the administered radioactivity
(84 to 97%) is eliminated in the feces, with unchanged diquat accounting for
at least 57$ of the original dose. About 4 to 11% of the 14c oral doses was
excreted in the urine within 48 hours after treatment, whereas biliary excra-
tion accounted for less than 5% of tne administered dose within 24 nours.
Oiquat monopyridone was identified in the urine and feces, and diquat dipyri-
done in the urine only.
A somewhat higher absorption rate was reported for dogs. Approximately 2y
to 32% of the orally administered dose was excreted in the urine. Absorbed
diquat appeared to preferentially accumulate in the kidney, but has also been
detected in heart, lung, liver, and adrenal tissue. After single oral doses Df
116 to 230 my diquat ion/teg body weight (bw), diquat concentrations in tissues
were generally less than 3 ug/g but were as nigh as 10 ug/g in the kidney after
4 hours. Residues in most tissues decreased during the period from 4 to 48
hours after the dose administration. In 8-week feeding studies with rats
(estimated dose of 12.5 mg diquat ion/kg/day), diquat concentrations in tissues
generally remained less than 0.001 ug/j.
1-1
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No data were obtained on levels of diquat in drinking water, from dietary
intake, or in ambient air. However, an interim tolerance of 10 ug/L was estaD- j
lished for drinking water residues, and tolerances in or on raw agricultural ;i
commodities and in foods have also been established. There are insufficient
:|
data to determine whether drinking water, food, or air is the major contributor '<
to total intake. :;
Diquat has moderate acute oral toxicity in mammals. Oral LOgo values for ;
various species were between >26 (for dog) and 430 mg diquat (for rat) ion/kg
:j
bw. The most notable effects of oral doses were an increase in gastrointestinal .'
water content and hemoconcentration. Diquat has a profound effect on body i-
water distribution; dehydration may play a key role in mortality.
Oral doses from 10U to 20U mg diquat ion/kg bw in rats caused minor histo-
pathological changes in the gastrointestinal (GI) tract, the kidney, and, to ;
some extent, tne liver.- The Lowest-Observed-Adverse-Effect Level (LOAEL) for a j
single oral dose in rats (judged by an increase in the water content of the GI
tract} was reported to be 18.4 mg diquat ion/kg bw. This was, however, the
lowest dose tested in that study. Distinct histopathological changes of the GI
tract and kidneys of monkeys that died following oral doses of 100 to 40U m^
diquat ion/kg were noted. No specific damage to lungs of laboratory animals
has been reported following oral administration of diquat.
No signs of irritation to digestive mucosa were observed when diquat was
administered in the drinking water to rats (500 and 1,000 my/L for 20 and 8
days, respectively) and rabbits (130 ana 50U mg/L for 6 and 10 days,
respectively).
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In a 4-week dietary study with male and female Charles River CD rats, a
No-Observed-Adverse-Effect Level (NOAEL) of 6.7 mg/diquat ion/kg/day was iden-
tified, Chronic feeding studies in rats and dogs have shown that cataract
formation is the most sensitive toxicological indicator of diquat exposure.
Cataract formation was found to be both dose and time dependent. A high fre-
quency of cataract formation was observed in rats following exposure for 2
years or longer to doses as low as 1.8 my diquat ion/kg/day. No-cataracts were
observed in dogs administered doses of 1.2 rag/kg/day for 4 years.
The mutagenic potential of diquat was studied in a number of bacterial and
eukaryotic systems. The results reported in the literature are contradictory.
Both positive and negative findings have been reported in the Salmonella assay,
unscheduled DNA synthesis, and mitotic gene conversion assay. While diquat
induced recessive lethal damage in Aspergillus. it failed to do so in Droso-
phila. There was no evidence to suggest that diquat is carcinogenic, No
antifertility or teratogenic effects were observed in mice, rats, or raooits
after oral diquat administration. However, teratogenic effects were observed
when diquat was administered to rats and mice via intraperitoneal (ip) or
intravenous (iv) injections.
A number of cases of diquat poisoning in humans were reported. In 10
cases of oral poisoning reported, 6 deaths occurred. Each individual probably
swallowed at least 15 ml Reglone (a dose containing 3 g diquat). The victims'
clinical signs indicated toxicity to the GI tract, brain, and kidney. In otner
cases, doses of about 5 mL fteglone (about 1 g diquat) were not lethal, but GI
tract and renal damage were observed.
The mechanism of diquat toxicity is not clear. One proposed mechanism
involves the reduction of dipyridylium cations to free radicals, which ;nay
1-3
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react with oxygen or cell constituents, leading to cell injury or deatn oy as
yet unknown pathways. Lipid peroxidation has been implicated in diquat-induced
tissue injury, but destruction of membrane lipids by auto-oxidation has not
consistently been found. The generation of oxygen-reactive species that accom-
panies diquat metabolism may play a role in the chemical's mode of action, out
the exact method by which these compounds act in association with diquat has
not yet been elucidated.
Since the principal acute effects of diquat toxicity appear to be related
to tissue dehydration and hemoconcentration, One-day Health Advisory (HA)
values were based on a study using water accumulation in the GI tract as an
endpoint. In this study, a dose of 18.4 mg/kg/day was identified as the LUAEL,
and the calculated une-day HA value for children is 20U ug diquat ion/L. No
studies were located that were suitable for calculation of Ten-day HA values.
The Longer-term HA value, discussed below, is recommended to serve as a consar*-
vative estimate of appropriate Ten-day HA values. A 4-1/2-montn sudcnronic
study in rats served as a basis for calculation of the Longer-term HA. This
study identified a dose of 1.1 mg/kg/day as a Low Effect Level (LEL). The
calculated Longertenm HA values for children and adults are 40 and 10U uy
diq.uat ion/L, respectively. The Reference Dose (RfD) and Drinking water
Equivalent Level (DUEL) were calculated based on a chronic study in rats using
cataract formation as an endpoint. This study identified a dose of U.22 m^/TJ/'-J
as a NUAEL, and the calculated rtfD and DUEL are 2.2 uy diquat ion/kg/day ana
3U uy diquat ion/L, respectively. Evidence from 5 studies sugyests that
Diquat may not be carcinogenic. -.owever, two of these oncogenicity studies in
rats and mice require additional :a:3 sefore a conclusion can be reacned. Con-
sequently, no carcinogenic ris< assess men: for diquat has been performed. No
previous guidelines or standares *:" J'il exposure to diquat were found.
1-4
i
il
II
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II. PHYSICAL AND CHEMICAL PROPERTIES
Qiquat is a dipyridylium herbicide that has been used extensively since
tne late 1950s to control Doth terrestrial and aquatic weeds. Diquat, marketed
in this country under the trade name Reglone (Vanholder et al., 1981), is
6,7-dihydrodipyrido(l,2-a:2',l'-c)pyrazdiium dibrornide (Akhavein and
Linscott, 1968). The chemical structure of diquat is presented in Table II-l.
Diquat dibromide is manufactured by reacting 2,2-bipyridyl, which is a
product of oxidative coupling of pyridine in the presence of heated Raney
nickel, with 1,2-dibromoethane in water (Worthing, 1979). The product is
crystallized from water as a pale yellow monohydrate (Windholz, 1983). Diquat
is stable and nonvolatile in the solid state and in neutral or acidic solutions
at ordinary temperature, but is volatile and decomposes in .basic solutions (pH
9 to 12) (Calderbank, 1968). Diquat is also photosensitive and is rapidly
decomposed by sunlight at ordinary temperatures. Diquat is highly soluble in
water and generally insoluble in nonpolar organic solvents. Solutions of
diquat are easily identified by their characteristic green color due to stable
free radical formation in alkaline solution. This property has been useful for
cnemical detection (Pasi, 1978). Diquat is commercially available in aqueous
concentrates of 140 and 200 g diquat ion/L (Vanholder et al., 1981). The
physical and chemical properties of diquat dibromide are summarized in Table
II-l.
Oiquat may be reduced to form a stable free radical (as shown in Figure
11-1} of a characteristic green color. The stability of the free radical is
attributed to the relatively large luioer of positive centers available to tne
odd electron (Akhavein and Linscott, H6d>. This reduction may occur in alka-
line solutions (pH 9 to 12) and in Dioloyical systems (Calderbank, 1968).
II-l
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Table Il-i. Physical and Chemical Properties of Diquat Dibromide
Property
Chemical Abstracts
Service (CAS)
Registry Number
Registry of Toxic
Effects of Chemical
Substances (RTECS)
Number
Synonyms
Chemical formula
Structure
Specific Gravity
Molecular weight
Valence state
Melting point
Vapor pressure
Absorption
Solubility in water
(w/v)
'
Value
85-00-7
JM569UOOO
Dipyrido(l,2-a:2M'-c)pyrazinediium, 6,7-dihydro-,
dibromide
Aquacide
Oeiquat
Oiquat
Ethylene dipyridylium dibromide
FB/2
Preeglone
Region
Reg lone
Reg 1 ox
Weedtrine-D
1,1-Ethylene 2,2-dipyridylium dibromide
5,6-Dihydro-dipyrido(l,2a:2,lc)pyrazinium dibromide ,
6,7-DThydropyrido(l,2-a:2',r-c)pyrazinedium di&romide!
9,10-Oihydro-8A,10A-diazoniaphenanthrene dibromide
[C12H12N2]Br2
1.22-1.27 (32U0C.
344.07
2+
Below 32U°C (decomp.). Also reported as 335 to 34U3C
Very low
UV max: 3U6.31 nm ( max 18,000)
70% at 2U'C
SOURCE: Adapted from Windholz (1983); Tatken and Lewis (1983).
II-2
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8-f V
tie.
Fmndicaf
Fiyure II-l. Reduction : j^ja: co the free radical
SOURCE: Adapted from A
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Diquat salts are strong electrolytes and are largely dissociated in water.
As a strong base, diquat readily undergoes exchange with cations of several
constituents of soil. This property causes diquat to be strongly adsorbed to
soil particles, particularly clay. Therefore, diquat is used to kill weeds
just before planting or within a few days of crop emergence. Additionally, it
can be used to kill weeds between rows, as long as none reaches crop foliage
(Calderbank, 1968; Pasi, 1978).
II-4
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III. TOXICUKINETICS
A. ABSORPTION
Oiquat appears to be poorly absorbed from the GI tract of rats. Clarsc and
Hurst (iy7u) reported that diquat is approximately 20 times more lethal in rats
when given by subcutaneous (sc) injection tnan by the oral route (see Chapter
V.A.I). The difference in toxicity after oral and parenteral administration
.suggests minimal absorption from the GI tract. Daniel and Gage (1966) admin-
istered a single oral dose (unless otherwise specified, all oral doses were
administered by gastric intubation) of either 14c-diquat dibromide (2.7 or 5.4
mg diquat ion/teg bw) or 14c-diquat dichloride (15.8 or 17.3 my diquat ion/icg
bw) in aqueous solution to male albino Wistar rats (200 to 230 g). No informa-
tion was provided on the number of animals per dose level or the age of the
rats. At the doses used, no obvious toxic effects were observed. Following
administration of the compound, 84 to 97% of the total radioactivity was
excreted in the feces, almost entirely within the first 2 days. Only 4 to li'i
of the total radioactivity was excreted in the urine. From these findings,
together with the absence of any marked biliary excretion, the authors con-
cluded that diquat was poorly absorbed from the gut. Similar findings were
reported by Mills (1976) who observed that 6% and 87% of a single oral dose of
4b my 14c-diquat ion/Icy bw administered to rats appeared in the urine and fe>w
respectively, within 4 days (mainly within the first 48 hours).
A somewhat higher level of diquat absorption was reported in dogs. When
1U to 15 my 14C-diquat dibromide was administered orally to dogs, 29 to 32% of
the initial labeled dose was recove-eo in tne urine and 51 to 62% in the feces
in the subsequent 3 days (ICI, I960'. !i :ne first 24 hours, 25 to 28% of trie
radioactivity appeared in the urine. Sennet: at al. (1976) reported that 10 to
III-l
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20% of a single oral dose of 0.012 mg diquat/kg bw was absorbed in 6 hours in
greyhound dogs.
Czyzewska et al. (1985) investigated the intestinal transport mechanism of:j
diquat; Various tissue segments, obtained from the small intestines of mongrel !
rabbits, weighing'2 to 3 kg, were bathed in aqueous solutions containing a final.;;
concentration of 1.0 mM diquat ion. At concentrations of 0.05 and 1.0 mM,
diquat did not pass through the intestinal wall to any significant degree. -'.\
.1
Diquat moved approximately two to three times faster in a portion of small
intestine devoid of both mucosal and serosal cells than in segments containing
one or both of these epithelial cell layers. Movement was slowest in an intact '.
i
segment of the intestine when diquat was applied to the mucosal side of the ;!
tissue. The presence of 1.0 mM ethyl alcohol reduced by 50% the passage of
diquat in samples lacking both serosa and miicosa. At 5.0 and 1.0 mM concen-
trations, respectively, alcohol moderately and slightly inhibited diquat pene- (
I
tration in serosa-free intestinal tissue.
The relatively slow movement of diquat into intestinal cells and the
virtual absence of intestinal diquat penetration at low concentrations was
attributed to the chemical's poor fat solubility and bivalent positive charge
and to the functional interdependence of the various epithelial layers of the
intestinal tract. The effect of alcohol appeared to be related primarily to
changes in the permeability of the subepithelial layers of the tissue. Overall,
the data support observations that diquat is poorly absorbed from the gut and
is readily eliminated in the feces.
III-2
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B. DISTRIBUTION
Rose et al. (1976) studied the tissue distribution of diquat in male
Alderley Park (Wistar-derived) rats administered a single oral dose of 680 umol
14c-diquat ion/kg bw (125 mg/kg). The age, weight, and number of the animals
were not reported. Animals were killed at 2, 4, 17, or 30 hours after treat-
ment, and organs were removed for analysis. The mean concentration (three or
four animals/determination) of diquat in the kidney was high (relative to other
organs) at all times measured and ranged from 23.8 nmol/g wet weight tissue .
(4.39 ug/g) at 2 hours after treatment to 48.4 to 54.0 nmol/g wet weight
tissue (8.92 to 9.94 ug/g) at 17 to 30 hours after treatment. Concentrations
of 6.1 to 13.9 nmol/g wet weight (1.12 to 2.56 ug/g) in the liver and of 7.5 to
16.0 umol/g wet weight (1.38 to 2.95 ug/g) in the adrenals were measured.
These levels were somewhat higher than that of the plasma (5.0 to 6.6 nmol/g
wet weight or 0.92 to 1.22 ug/g). There was no evidence that any other organs
accumulated diquat to the extent that was observed in the kidney, jr^ vitro
studies showed no significant accumulation of diquat in rat tissue slices of
various organs incubated in a medium containing 10-6 M (0.184 mg diquat ion/L)
14c-diquat, except for kidney tissue. The rate of uptake by kidney slices was
rapid and was complete by 1 hour.
In a similar study, Kurisaki and Sato (1979) measured levels of diquat in
tissues of 13 male Wistar rats given a single oral 1050 dose of 231 mg diquat
ion/kg bw (administered as Reglox containing 30% diquat dissolved in water).
Two hours after dosing, the heart contained approximately 12 ug diquat ion/g
tissue, and the lungs contained 7 ug diquat ion/g tissue. At 24 hours, the
concentration in the heart'had decreased to about 2 ug/g tissue, and the Tuny
content nad decreased to 5 ug/g of tissue. These values changed little by *3
III-3
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nours. The diquat concentration in the kidney tended to increase from 24 to 48
hours after dosing. After an oral dose of half the LD50 (116 nig/kg bw) to five
additional rats, a low concentration of diquat was detected in all organs
(generally.<2 ug/g at 2, 5, and 9 days after treatment) except the kidney,
which had a slightly higher concentration on the second day only.
C. METABOLISM
As previously noted, Daniel and Gage (1966) found that 84 to 97% of an
oral dose of 14f,-diquat given to male albino Wistar rats was recovered in the
feces. Most of this radioactivity (70% of the original dose) was in the fonti
of degradation products. The authors concluded that this degradation was due
to microbiological action within the intestine, since incubation of diquat with
a fecal homogenate resulted in 40 to 50% destruction of diquat within 24 hours,
whereas incubation with a heated fecal homogenate resulted in only a minor loss
of diquat in the same period. There also may have been some gastrointestinal
absorption of degradation products formed within-the gut, since metabolites
detected in the urine after oral diquat administration were not observed after
sc administration of the herbicide.
The chemical nature of these metabolic products was studied by Mills
(1976). Five male albino Wistar rats of the Alderley Park strain, weighing 130
to 2UU g, were administered a single oral dose of 45 mg HC-diquat ion/kg bw
(as an aqueous solution of the dibromide salt). The age of these animals was
not reported. In contrast to the findings of Daniel and Gage (1966), unchanged
diquat was found to be the major radioactive component of both urine (5i of tf
dose) and feces (at least 57% of tne -aose). Analysis of urine samples by
chromatography .showed the presence tf :-o wcaoolites: 0.2% of the dose was
excreted as diquat monopyridone and J.I*, as jiquat dipyridone. Diquat
II1-4
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monopyridone (4,3% of the dose) was identified as the major metabolite in the
feces. fliquat dipyridone was not detected in the feces. ^n vitro studies
showed that rat cecal microflora could metabolize about 10% of added diquat,
with the formation of some diquat monopyridone. Mills concluded that there was
good evidence that diquat was metabolized only to a limited extent by the .gut
microflora and by the body tissues but attributed the difference between these
results and those of Daniel and Gage (1966) to differences in. the extraction
procedures used.
D. EXCRETION
Most of the diquat administered to rats was eliminated in the feces, witn
11% of the dose or less eliminated in the urine. Daniel and Gage (1966) demon-
strated that over 80% of a single oral dose of l^C-diquat (administered as
diquat dibromide or diquat dichloride) was excreted in the feces of rats within
2 to 3 days (see Section III.A). Excretion of the dibromide compound appeared
to be complete within 2 days. There was no indication that the nature of the
anion influenced excretion.
Absorbed diquat is largely eliminated by the kidneys, with some biliary
excretion. Daniel and Gage (1966) observed that 4 to 1U of a single oral
dose of diquat dibromide (2.7 or 5.4 mg diquat ion/kg bw) or diquat dichloride
(15.8 or 17.3 my diquat ion/kg bw) was excreted in the urine of rats witnin
48 hours after treatment. These findings are in agreement with the results of
other investigators.- Lock and Isnmael (1979) observed that 7.5% of a single
oral dose of diquat (125 mg ion/kg bw) in male Alderley Park (Wistar-derived)
albino rats was excreted by the kidneys during the first 24 hours, while
litchfield et al . (1973) reported tnat 5.5% of a single oral dose of diquat
III-5
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dibromide (60 my diquat Ion/kg bw) was excreted in the urine of male rats (of
the same strain) within 7 days.
Daniel and Gage (1966) found biliary excretion of diquat in rats to be
less than 5% of the dose within 24 hours after oral administration of HC-
diquat dibromide at levels of 0.64 or 34.3 my diquat ion/kg bw. Similar values
for biliary excretion were reported by Hughes et al. (1973) for female Wistar
albino rats, female English guinea pigs, and female Dutch rabbits given 1*C-
diquat dichloride intraperitoneally (7.36, 2.39, and 2.39 mg diquat ion/kg bw,
respectively). Biliary excretion of diquat, in 3 hours, was less than 5X in
these animals. Excretion of 14c in urine, within 3 hours, was 82, 45, and 64*,
of the dose for rats, guinea pigs, and rabbits, respectively.
E. BIOACCUMULATION AND RETENTION
Litchfield et al. (1973) fed 40 rats (Alderley Park, Wistar-derived, mala
and female, 210 to 240 g; age not specified) a diet containing diquat dibromide
monohydrate at a level of 250 my diquat ion/kg feed. This represents an esti-
mated dose level of 12.5 mg diquat ion/kg/day, assuming 0.4-kg rats consume
20 g food/day. After 2, 4, and 8 weeks, 10 treated and 5 control animals were
sacrificed, and major organs were removed for tissue analysis. Tissue concen-
trations of diquat in the kidney, brain, liver, lung, stomach, and small and
large intestines were generally less than 1 ug/g, with preferential accumula-
tion initially in the stomach and large and small intestines and later in the
kidney and large intestine. Concentrations of diquat in the kidney at 3 weeks
were approximately five tine's those determined at 2 and 4 weeks. Within 1 week
of return to a normal diet, no diqjat *as detectable in any tissue (detection
limit of Q.05 ug/g).
III-6
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F. SUMMARY
In rats, about 4 to 11% of oral doses of diquat dibromide (2.7 or 5.4 mg
diquat ion/kg bw) or diquat dichloride (15.8 or 17.3 mg diquat ion/kg bw) was
absorbed from tne gastrointestinal tract and excreted in the urine of rats
witnin the first 48 hours after treatment. About 80% of the dose of diquat *as
excreted in the feces. Biliary excretion in rats following an oral dose was
less than 5% of the administered dose within 24 hours.
-Absorption of oral doses was somewhat greater in dogs than in rodents.
ADout 3U percent of tne oral dose of HC-diquat dibromide (10 to 15 mg/dog)
was recovered in the urine 3 days after dosing. Radioactivity eliminated in
the feces accounted for 51 to 62% of the dose.
In studies with rats, unchanged l*C-diquat was found to be the major radio-
active component of both the urine (5% of the original dose) and feces (at
least 57% of the original dose). Metabolites identified in the urine were
diquat monopyridone (0.2% of tne dose) and diquat dipyridone (0.1% of tne
dose). The major metabolite in the feces was diquat monopyridone (4.3% of .tne
dose).
Following absorption, diquat appeared to be relatively uniformly distrio-
uted among organ tissues in rats receiving doses of 116 to 125 mg diquat ion/:
-------
concentration in the kidney increased between 24 and 48 hours. _In vitro
studies showed that diquat accumulates in the kidney, but not in other tissues,
An 8-week feeding study in rats demonstrated that diquat does not substan-
tially accumulate in animals administered 250 my diquat ion/ky feed in their
diet (estimated daily dose of 12.5 mg diquat ion/kg DW). Oiquat tissue levels
were generally less than 1 ug/g in the brain, liver, lung, stomach, and.small
, and large intestines. Concentrations in the kidney and large intestine
increased and rose above the 1 ug/g level during the latter part of the 3-week
feeding period. No diquat was detectable in any tissue within 1 week after
return to a control diet.
in-a
-------
IV. HUMAN EXPOSURE
Humans may be exposed to chemicals such as diquat from a variety of
sources, including drinkiny water, food, ambient air, occupational settings,
and consumer products. This analysis of human exposure to diquat is limited
to drinking water, food, and ambient air because these media are considered to
s
be sources common to all individuals. Even in limiting the analysis to these
three sources, it must be recoynized that individual exposure wi-11 vary widely
based on many personal choices and on several factors over which little control
exists. Daily exposure and intake are profoundly affected by the area in which
an individual lives, works, and travels; a person's diet; and physiologic
characteristics related to aye, sex, and health status. 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 diquat in the
environment has been presented in an interim draft report by Johnston et al.
(1984). The section that follows summarizes the pertinent information presented
in that report to assess the relative source contribution of diquat from drintc-
ing water, food, and air.
In Section A, Exposure Estimation, available information is presented on
the range of human exposure and intake for diquat from drinking water, food,
and ambient air for a 70-kg adult male. It is not possible to provide an
estimate of the number of individuals experiencing specific combined exposures
from these three sources.
IV-1
-------
A. EXPOSURE ESTIMATION
1. Prinking Water
No data were obtained on levels of diquat in drinking water. However, an
interim tolerance of 10 ug/L was established for residues of diquat in drinking
water resulting from the use of its dibromide salt to control aquatic weeds in
canals, lakes, ponds, and other potential sources of drinking water (U.S. EPA,
1979). The maximum intake of diquat from drinking water following this use was
estimated. Assuming that a 70-rkg adult consumes 2 liters of water per day, a.
maximum intake of 0.29 ug/kg/day was calculated* The level of diquat in most ;
drinkiny water may be considerably lower than levels calculated here since
this calculation assumes maximum water residue tolerance exposure. The compound !
has also never been reported in drinking water. In addition, .the value presented'
does not account for variances in individual exposure or uncertainties in the :;
assumptions used to estimate exposure.
2. Diet
No data were obtained on the dietary intake of diquat in the United States. !,
Tolerances for diquat in and on raw agricultural commodities and in foods are it
listed in Table IV-1. These data cannot be used, however, to estimate typical
dietary intake.
3. Ai£
p
No data were obtained on levels of diquat in ambient air. Therefore, the
intake of diquat from ambient air could not be estimated.
IV-2
-------
Table IV-1. Tolerances for Diquat in or on Agricultural Products
Commodity Tolerance (ug/kg food)
Food
Potatoes, processed* 50
Raw agricultural commodity
Cattle
fat 20
meat by-products 20
meat 20
Eggs 20
Goats
fat 20
meat by-products 20
meat 20
Hogs
fat 20
meat by-products 20
meat 20
Horses
fat 20
meat by-products 20
meat 20
Milk 20
Potatoes 10U
Poultry 20
fat 20
meat by-products 20
meat
Sheep
fat 20
meat by-products 20
meat 20
Sugarcane 50
^Including potato chips.
SOURCE: Adapted from U.S. EPA (19di).
IV-3
-------
-------
B. SUMMARY
Data on the intake of diquat 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.
IV-4
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V. HEALTH EFFECTS IN ANIMALS
A. SHORT-TERM EXPOSURE
1. Lethality .
Data on the acute oral toxicity of diquat dibromide or diquat dichloride
in various animal species are summarized in Table V-l. Values for an oral
ranged from >26 to 433 mg diquat ion/kg bw, and most were within the range of
100 to 200 mg diquat ion/kg bw. LDso values following subcutaneous administra-
tion were 11 to 12 my diquat ion/kg bw (Clark and Hurst, 1970). These results
show that diquat has moderate acute oral toxicity, and the potency of diquat
when administered subcutaneously is much greater than when administered orally.
This may be due to poor absorption of diquat from the GI tract.
Deaths of animals following acute oral administration of diquat occurred
over several days. Death in rats following oral doses of diquat dichloride
(900 umol or 166 mg diquat ion/kg bw) occurred during the first 9 days after
treatment, although about 50% of these occurred within the first 3 days
(Crabtree et al., 1977). Similarly, all four deaths in a group of :monkeys
treated orally with diquat dichloride monohydrate (100, 300, or 400 mg diquat
ion/kg bw} occurred within 4 days of dosing (Cobb and Grimshaw, 1979). Deatns
in cattle following oral doses of diquat dibromide (30 to 100 mg diquat ion/kg)
occurred between 15 hours and 15 days.
In a study by Crabtree et al. (1977), a single LQ$Q dose of 900 umol (166
mg) diquat ion/kg bw (as diquat dichloride} dissolved-in sterile physiological
saline was administered orally (2 mL/ky by stomach tube) to 45 male Alderley
Park (Wistar-derived) rats. The body weignt range of the animals was 15U to
2UU g; age was not specified. In the initial 24 hours after dosing, the annals
V-l
i
-------
Table V-l. Acute Oral Toxicity of Oiquat in Several Animal Species
Compound
Oiquat
dibromide
Diquat
di chloride
Species
Mouse
Rat
Rat
Rat
Rat
Rat
Rat
Rabbi t -
Rabbit
Dot]
Dog
Dog
Guinea pig
Chicken
Cattle
Cattle
Monkey
House
Rat
u$o
(mg diquat
ion/kg bw)
125
166
121-147
214-337
231
215-235
321-433
100
101
100-2003
>26
approx. 107
approx. lOOa
215-430
30
30*
lOOb
125
3U2
Reference
Clark and Hurst (197U)
Crabtree et al . (1977);
Lock (1979)
Gaines and Under (1986)
Plant Protection Ltd.
(I960)
Clark and Hurst (1970)
1C I (1962, 1982)
Raven (1981)
ICI (1960, 1982)
Clark and Hurst (1970)
Clark and Hurst (1970)
ICI (1960)
ICI (1982)
Clark and Hurst (1970)
ICI (1960, 1982)
Walley (1962)
Clark and Hurst (1970)
Cobb and Grimshaw (1979)
ICI (1963)
ICI (1962)
aEstimated by Clark and Hurst (1970).
V-2
-------
appeared relatively normal except that their water intake was only one-fifth
that of the control animals. After 24 hours, however, the authors reported
that tne rats were lethargic, exhibited signs of piloerection, and began to
excrete feces of a mucoid, ropey form with a characteristic greenish color.
Clark and Hurst (iy?U) reported that this color was due to the reduction of
diquat during bacterial metabolism.
Crabtree et at. (1977) also noted that animals dying in the first 3 days
after oral dosing did not show weight gain in the first 24-hour period (98.4 _*
0.6% of original weight, p <0.01). However, a weight gain at 24 hours was
reported for animals that survived for at least 3 days after the test {103.3 +_
1.1% of original weight, p <0.01). Animals that did not gain weight 24 nours
after an oral LDgQ dose had significantly greater water content in the GI tract
than control rats for up to 8 days and significantly more pronounced hetiocon-
centration than the animals in the control group (p <0.05). The authors attri-
buted deatns occurring shortly after dosing (within the first 3 days) to rapid
fluid loss into the lumen of the GI tract. Later deaths (occurring between 4
and 9 days following dosing) were unexplained, although the authors suggested
that prolonged dehydration was at least partially responsible.
Single intragastric doses of 0, 160, 241, 35U, 535, or 963 mg diquat
ion/kg bw (as diquat dibromide) were administered to groups of five fasted male
(2UB to 269 g) and female (201 to 226 g) Sprague-Oawley-derived rats (Raven,
19ai). The rats were observed for signs of toxicity including depression,
decreased food consumption, diarrhea, and death. Depression and decreased food
consumption were observed at all dose levels. In general, no gross pathologi-
cal changes were seen that could be attributed to the herbicide. The mortality
at dose levels 0, 160, 241, 353, 535, and 963 mg diquat ion/kg bw was. 0/5, 0-5.
i
V-3
-------
0/5, 1/5, 4/5, and 5/5, respectively, among male test groups and 0/5, 0/5, 0/5,
5/5, 5/5, and 5/5, respectively, among fanale test groups. Most of the deaths
occurred within the first 5 days. 105^^95% confidence limits were 0.31 (0,44
to 1.5) g/kg in males (equivalent to 433 my diquat ion/kg), and 0.6U (0.31 to
1.2) g/kj in females (equivalent to 321 mg diquat ion/kg).
Clark and Hurst (1970) reported that Alderley Park albino rats surviving
1 year after receiving one near-lethal oral dose of diquat exhibited, no addi-
tional toxic effects attributable to diquat exposure other than the immediate
short-term effects described previously.
Cobb and Grimshaw (1979) administered single oral doses of 100, 200, 300,
or 40U m»j diquat ion/ky bw (as the dichloride monohydrate in aqueous solution)
to male cynomolgus monkeys. No information on age or body weight of the ani-
mals was provided. The animals were observed for 14 days following treatment.
Death occurred in 1/2, 0/4, 1/2, and 2/2 of the monkeys dosed with 1UO, 20U,
300, or 400 mg diquat ion/leg bw, respectively. All deaths occurred within 4
days. Within the first 12 hours, all monkeys in this study exhibited diarrhea
and vomiting, and 5 of the 10 monkeys became lethargic and collapsed. Samples
of blood and urine were taken at 4, 24, 48, and 72 hours, and 4, 7, and 14 days
post-treatment. Increases in serum urea, plasma glucose, serum glutamic-oxalo-
acetic transaminase (SGOT), and serum glutamic-pyruvic transaminase (SGPT) were
noted in almost all animals witnin 24 hours compared to pretreatment levels.
These values remained elevated in the monkeys that subsequently died; surviving
animals snowed a return to normal values between 4 and 14 days after dosing.
Analysis of urine samples of almost all the animals showed a reduced-pH and an
increase in protein, glucose, total -educing substances, blood pigments, and
red blood cells 24 to 48 hours after dosing. These values in surviving animals
V-4
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returned to normal 4 to 14 days after treatment. Histological examination of j
the animals that died during the study revealed large areas of necrosis and j
exfoliation of the epithelium in the stomach and.intestine. The kidney was also
severely affected, with necrosis and exfoliation of epithelial cells in both the .
proximal and distal tubules. Only minimal histopathology was observed in the
i
liver. The authors concluded that death arose primarily from the destructive
effects of diquat on the epithelial cells of the GI tract and the renal tubule.
Single oral doses ranging from 20 to 100 mg diu.uat/kg bw were administered
to cattle (Walley, 1962). It was reported that the herbicide {Reglone) caused
the cattle to grind their teeth and made them "dull and miserable." Once the
cattle were recumbent, they were not responsive to external pain stimuli. In
higher dose groups (30 to 100 mg diquat/kg), deaths occurred between 15 hours
and 15 days following treatment. Postmortem examination showed patchy hemor-
rhage in the omasum and abomasum, destruction of mucosa and hemorrhage in the
duodenum, and green coloration in the posterior portion of the colon. Hemor-
rhage was also seen in the heart, kidneys, and bronchi. The LOsg for cattle
was about 30 mg diquat ion/kg bw.
2. Other Effects
The signs observed following oral administration of an acute lethal dose
of diquat do not suggest an obvious mode of action. Acute toxic effects noted
in rats included gross abdominal swelling, muscular twitching, and an erratic
gait with splayed hind limbs (ICI, 1962).
a. Gastrointestinal tract
Physiological and/or hi-stoloyi :ai -manges in the GI tract have been
reported following oral administration of diquat in rats (Crabtree et al.,
V-5
-------
1977; Crabtree and Rose, 1978); monkeys (Cobb and Grimshaw, 1979); and cloys
and rabbits (ICI, 1960). In a study by Crabtree et al. (1977), a single oral
L05Q dose of 900 umol (166 nig) diquat ion/ky bw to male Alderley Park (Wistar-
derived) rats produced a rapid and significant increase in the water content of
the lumen of the GI tract, an effect that was accompanied by significant hemo-
concentration. This response peaked at 24 to 48 hours after dosing. The changes
in fluid accumulation in the gastrointestinal tract which occurred at 24 hours
were related to the dose of diquat administered. By the eighth day, no difference
between control and diquat-treated groups could be detected.
In another experiment by Crabtree et al. (1977), 5 to 15 rats/yroup were
treated with single oral doses of approximately 0, 100, 350, 600, 900, or 1,21)0
umol (0, 18.4, 64.5, 111, 166, or 221 mg, respectively) diquat ion/kg bw. The
LOAEL for an increase in water content of the GI tract within 24 hours was 13.4
mg diquat ion/kg, bw, the lowest dose tested. It was not noted whether this
increase in water content was statistically significant. At the 900 umol dose,
the water accumulation in the lumen of the GI tract was accompanied by reducad
urine output and a significant decrease (p <0.05) in the water content of the
blood, liver, and muscle. Only minor histological changes in the GI tract were
observed after oral administration of diquat. The most frequent observations
were edema of the submucosa of the stomach at the junction of the glandular and
nonglandular regions and a slight to moderate dilation of the lacteal and
submucosal lymphatic vessels of tne small intestine and cecum. Fluid accumulation
in the gastrointestinal tract following oral diquat administration appeared to
be dose-related and was accompanied ay a decrease in the water content of other
tissues, particularly blood. A_cor-eU:ion was established between deaths
occurring within the first 3 days 3? an or*; iDgQ and fluid redistribution;
those rats maximally affected (no -ei^nt jain) had significantly (p <0.01) none
V-6
-------
water in the gastrointestinal tract and significantly (p
-------
monkeys administered diquat. In animals that died following a single oral dose
of 100, 300, or 4UO my diquat ion/kg bw, larye areas of the stomach, small
intestine, and large intestine showed evidence of necrosis and exfoliation of
t.ie epithelium that exposed the underlying submucosa. The most severe damage
occurred in the villi, although the bottom of the crypts frequently were
unaffected. The remaining intestinal tissue was infiltrated with mononuclear
cells, and the submucosa was hyperemic. The authors concluded that death in
the dosed monkeys resulted from the effects of the destruction of the epitne-
lial lining of the GI tract combined with extensive kidney damage.
In dogs and rabbits, the destruction of the gastric mucosa observed after
a single oral LDsy dose of diquat dibrornide was followed, in some instances, by
perforation of the stomach wall (ICI, 1960).
b. Kidneys
Studies by Lock (1979) showed that a single oral dose of diquat produced
marked changes in renal function in the rat. These changes were attributed to
altered renal hemodynamics, rather than to direct renal pathological changes,
since only mild renal tubular damage was found on histological examination.
Forty-two fasted male Alderley Park (Wistar-derived) albino rats weighing 150
to 180 g were given a single oral dose of 540 umol (99 mg) diquat ion/kg bw.
The age of the animals was not specified. Twenty-four hours after treatment,
urine flow was significantly reduced, and there was a significant decrease in
clearance of urea, inulin, ja-amino-hippurate -(PAH), N-methylnicotinamide (NMN),
and diquat by the kidney. Measurements of plasma and red cell volumes in five
rats dosed witn 680 umol (125 mg) or 90U umol (166 mg) diquat ion/kg bw showed
hemoconcentration, which was caused Dy a significant reduction in plasma volume
without a concomitant alteration in red cell volume. The decrease in plasma
V-8
-------
volume was accompanied by a significant reduction in renal plasma flow only at
the 166 mg diquat ion/kg (IDso) level and was assumed to be due to a large
fluid shift from the tissues into the lumen of the GI tract. The author con-
cluded that diquat probably did not have a direct effect on the kidney.
Effects on renal hemodynamics were attributed to a reduction in plasma volume.
A separate study by Lock and Ishtnael (1979) confirmed the absence of a
direct effect of diquat on the rat kidney. Ten fasted male Alderley Park
(Wistar-derived) albino rats (150 to 180 g; age not specified) were each
treated orally with a single dose of 680 umol (125 mg) diquat ion/kg bw. At 5
to 24 hours after dosing, there was significant proteinuria and glucosuria and
an increase in the rate of exfoliation of renal tubular cells. Only mild,
focal hydropic degeneration of the proximal convoluted tubules was observed in
the kidneys upon histopathological examination 24 hours after treatment.
Studies using renal cortical slices showed that the activity of the pentose
phosphate pathway and fatty acid synthesis were not affected 24 hours after j
treatment, indicating that the redox state had not been altered. The limited
histological changes contrasted with the very marked effects that diquat had on
renal excretory function, thus providing further support to the hypothesis tna: ;
the reduction in renal function was primarily a result of fluid redistribution.
In contrast to the minimal pathological changes observed in the rat kidney, :
renal tubular necrosis was found to be one of the principal lesions of diquat
poisoning in cynomolgus monkeys (Cobb and Grimshaw, 1979). In animals that
died after receiving a single oral dose of 100, 300, or 400 mg diquat ion/ka
bw, severe exfoliation of the epithelium of the proximal and distal convoluted
tubules was observed upon histological examination. This change, which was
most severe in a monkey that died 24 nours after receiving 400 mg/kg, par-
V-9
-------
tidily extended along the collecting ducts. In animals dying on days 3 and 4,
the effects were less severe and the lesions appeared to be regressing.
c. Liver
Normal histology and normal serum enzyme levels indicated that liver necro-
sis was not present in Sprague-Daw!ey rats (survival time * 80 ^ 12 minutes)
administered an intraperitoneal dose of 230 umol (42 mg) diquat ion/kg as
diquat dichloride (Burlc et al., 1980). However, when Rose et al. (1974) admin-
istered diquat (as diquat dichloride) intraperitoneally to male Alderley Parx
(Wistar-derived) rats (weighing 180 to 220 g) at a level of 20 mg diquat ion/kg
bw, depletion of liver glycogen during starvation was found to be partially
prevented. Liver glycogen was 59% depleted after 24 hours of starvation in
treated animals, in comparison with a 99% reduction in saline-treated and
starved control rats. There was also a rapid and dramatic increase in blood
glucose levels in treated animals, and the authors suggested that the synthesis
of liver glycogen was stimulated as a result of diquat dosing. Glucose levels
returned to normal after approximately 7 hours. In contrast, treatment of
adrenalectomized animals with diquat resulted in a rapid loss of liver glycogen
and only a smaller increase in blood glucose. This led the authors to conclude
that elevation of blood glucose and control of liver glycogen utilization
following diquat administration was mediated by the adrenals.
Diquat appears to affect another hepatic metabolic system, lipid peroxida-
tion. Lipid peroxidation is associated with the selenium-containing enzyme
glutathione peroxidase, whose activity is very low in the liver of selenium-
deficient animals. A single Intrapericoneal injection of diquat (19.5 umol or
3.6 mg/kg DW) into selenium-deficient nale Holtzman rats caused rapid and
massive liver necrosis (Burk et al., 198U). A marked increase in lipid peroxi-
v-id
-------
dation, as measured by ethane production rates and by the appearance of malon-
aldehyde in the serum, accompanied the liver injury; SGPT activity was greatly
elevated 90 minutes after diquat administration, and death occurred within 2
hours. The authors hypothesized that since minimal lipid peroxidation and no
liver necrosis were detected in diquat-treated control rats (see above), lipid
peroxidation might be the biochemical, process responsible for the observed
diquat hepatotoxicity in selenium-deficient animals. This subject is further
discussed in Chapter VII, Mechanisms of Toxicity.
Histological examination of monkeys that died after receiving a lethal
oral dose of 100, 300, or 400 mg diquat ion/kg revealed minimal evidence of
liver toxicity (Cobb and Grimshaw, 1979). Only sinusoidal congestion and
scattered necrosis of single hepatocytes, with some hepatocyte vacuolization,
were observed.
d. Lungs
The effects of diquat on the lungs of rats and mice were evaluated follow- .'
ing, parenteral (Coulombe et al., 1984) and intratracheal (Manabe and Ogata, ;:
1986) administration. "
Coulombe et al. (1984) reported lung alveolar toxic damage in Swiss- ;|
Webster mice treated intraperitoneally with diquat at doses of 0.4, 4.0, and 40
my/kg. Three days after administration of diquat, a rise in the number of ;.
polymorphonuclear (PMN) cells per pulmonary surface unit was found that
increased linearly with the administered dose. A dose-dependent relationship
was observed in the inflammatory and epithelial regeneration component of tne
alveolar primary reaction. .;
V-ll
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Manabe and Ogata (1986) evaluated diquat toxicity ii adult male Fischer-
344 rats following intratracheal administration of the herbicide at doses of
2.43 x 10-2, 9.72 x 10-2, 3.89 x 10-1, OP 1.56 umol diquat/luny. The lunys of
rats injected witn lower doses of diquat (2.43 x 10-2 or 9.72 x 10-2 umol/luny)
showed only minimal or slight damage. Moderate alveolar damaye was observed at
a dose of 3.89 x 10-1 umol/luny. The most severe lung damage (severe hemorrnaye
and alveolar wall disruption) was observed at the highest dose (1.56 umol/luny).
3.. Subacute Toxicity
Oe Lavour et al. (1979) investigated the irritant effects of diquat on trie
digestive mucosa of rats and rabbits. Diquat was given in drinking water to
male rats at concentrations of 5UO and 1,000 mg/L for 20 and 8 days, respec-
tively (approximately 37.5 and 75 mg/kg/day, respectively, assuming a final
body weight of 4UO g and water consumption of 30 ml/day), and to male rabbits
at concentrations of 1UO and 500 my/L for 6 and 10 days, respectively (approxi-
mately 6 and 30 mg/kg/day, respectively, assuming a final body weight of 5 icy
and water consumption of 3UU mL/day). No signs of irritation of the diyestive
mucosa were observed in either rats or rabbits.
Chevron Chenical Company (1981) evaluated the toxicity of diquat dibromide
in rats fed diets containing the herbicide for 4 weeks. Five groups of 10 -nale
and 10 female Charles River CD rats of the Sprague-Oawley strain were fed Siats
containing 0, 75, 200, 350, and 500 ppm diquat ion. The overall means (expressed
as mg diquat ion/kg/day) of weekly intakes during 4 weeks of administration,
the 75-, 200-, 350-, and 500-ppm di^uac ion yroups were 6.7 (males « 6.1,
females » 7.2), 17 (males = 16.1, finales = 17.8), 29.6 (males » 27.8, females
= 31.4), and 39.7 (males = 37.7, fe-ules » 42.0). respectively. Reduced
gain, reduced food intake, and liver involvement were observed in animals
V-12
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receiving 200, 350, and 500 ppm diquat ion. Liver involvement was indicated
by low SGPT activities and hiyh serum cholesterol levels in these groups.
These effects were not observed in the 7S-ppm diquat ion group. Gaseous distan-i
sion of the cecum was observed in rats from all dosed groups. However, histo-
pathological examination did not reveal any morphological changes, and the
authors suggested that this observation is of doubtful significance. From the
results of this study, 75 ppm diquat ion (6.7 mg/diquat ion/kg/day) appears to
be the NOAEL in rats.
B. LONG-TERM EXPOSURE
1. Subchronic Toxicity
In the study oy Bainova (1969), rats (strain not reported) were administered
diquat orally for 4-1/2 months at 2.1 and 4.3 mg/kg bw/day (1.1 and 2.3 mg
diquat ion/kg bw/day). Biochemical changes were noted at both doses; the higher (t
dose produced more severe effects. Biochemical alterations included elevated
values of hemoglobin, erythrocytes, leukocytes and catalase activity. Cholines-
terase activity was reduced in cerebral homogenates. Histological examination
revealed ulcerouspurn lent tracheitis, inflammatory infiltration of the peri-
bronchial connective tissue, and hyperplastic peribronchiolar lymphatic tissue,
A papillomatous proliferation of the bronchiolar and bronchial epithelia was
noted. In the liver, a slight parenchyma! dystrophy of the hepatocytes and
hyperplasia of the Kupffer cells was produced. The testes of treated rats
exhibited mild edema of the interstice with expansion of the interstitial
space; the number of cells in the canals was reduced and there were almost no
spermatozoids in the majority of them. The histological changes were seen
in all test animals, however, the severity was greater at the higher dose.
i
V-13
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2. Chronic Toxlclty
Pine and Rees (197U) described the pathological changes occurring during
the development of lens opacities in diquat-fed rats. Wistar-strain albino
rats, 4 to 6 weeks old, were fed an ICI Alderley Park diet containing 0.05 or
O.U75% diquat dibromide (approximately 13 or 19 mg diquat ion/kg/day, assuming
a final body weight of 0.4 kg and daily food consumption of 20 g) for a period
of 7(J weeks. Information on sex, body weight, and number of rats per dose
level was not reported. Observations using a slit lamp and ophthalmoscope
revealed irregular opacity changes in the posterior cortex of the eye that
required 4 to 8 months to develop. A clearly defined cataract represented the
next stage and was followed by complete opacity. The development of lens
opacities resembled that caused by X-irradiation, although no evidence of
damage to the nuclei of the lens epithelial cells was observed. .The authors
concluded that similar cataracts are unlikely to occur in humans, since a
prolonged exposure to diquat is required.
Colley et al. (1985) evaluated diquat dibromide for potential carcinogeni-
city and chronic toxicity in a 2-year feeding study with rats. Charles River
CD rats of the Sprague-Oawley strain (five groups of 60 males and 60 females)
were fed a diet containing 0, 5, 15, 75, and 375 ppm diquat ion for 104 weeks.
The intake of diquat ion was calculated from the group mean estimated midweek
body weight and group mean food consumption data. The overall means (expressed
as mg diquat ion/kg bw/day) of weekly intakes during 104 weeks of treatment for
the 5-, 15-, 75-, and 375-ppm diquat ion groups were 0.22 (males » 0.19,
females = 0.24), 0.65 (males = 0.53, females = 0.72), 3.28 (males *-2.9l,
females * 3.64), and 17.16 (males » U.88. females =.19.44), respectively;
Dose-related cataractogenic effects were observed in males and females
V-14
-------
receiving 75 ppm and 375 ppm. There was weak evidence of tnis effect at 15 ppm
(0.65 mg diquat ion/kg bw/ a day) which was identified as the LOAEL. The
authors concluded that the NOAEL for cataract formation is close to 5 ppm (0.22
mg diquat ion/kg bw/day). The incidence of mortality was not affected by the
treatment. A reduction in appetite was recorded for rats receiving 375 ppm.
't
No toxicological ly significant changes were noted (in hematology, blood chemistry;;
urinalysis, histology, and gross pathology) other than reduction in hemoglobin
levels in the 75-ppm group and reduction in mean cell volume in the 75- and
375-ppm groups. However, several.minor variations were noted.
Rogerson and Broad (1978) reported a slightly higher no-effect level for
cataract formation in rats fed diquat dibromide for a period of 2 years.
Groups of 35 male and 35 female Wistar-derived rats (7 weeks old, 95 to 110 y) ij
from the Alderley Park colony were maintained for 2 years on diets containing
0, 15, 25, or 75 ppm diquat ion (approximately 0, 0.75, 1.25, or 3.75 mg di^jat
ion/kg/day, respectively, assuming a final body weight of 0.4 kg and food con-
sumption of 20 g/day). No statistically significant increase in cataract inci-
dence was observed in rats fed 15 or 25 ppm (0.75 or 1.25 mg diquat ion/kg/day,
respectively). Approximately one-third of the rats treated at 75 ppm (3.75 nig
diquat ion/kg/day) showed a statistically significant increased induction of
cataract formation at 9 months. The effect became marked by the second year;
approximately two-thirds of the animals at this dose level showed cataracts.
Based on this 2-year feeding study with rats, the authors concluded that the
NOAEL was 25 ppm diquat ion (1.25 mg diquat ion/kg/day), and the LOAEL was 75
ppm diquat ion (3.75 mg diquat ion/'
-------
the rat study, 25 male and 25 female Alderley Park albino rats,' weighing 180
to 200 g, were fed diets containing 0.001, 0.005, 0.01, 0.025, 0.05, or 0.15
diquat dichloride for 2 years. Assuming a final body weight of 0.4 kg and
food consumption of 12 g/day (Arrington, 1972), these doses would approximately
correspond to 0.22, 1.1, 2.2. 5.5, 11, or 22 mg diquat ion/kg/day, respectively.
Neither the number of animals per dose level nor the aye of the rats was speci-
fied. No mortality was observed. Food consumption was reduced, and some
reduction in growth rate occurred in animals receiving 0.1% diquat (22 mg
diquat ion/kg/day). Diets containing less than 0.1% diquat had no measurable
effect on body weight or food consumption. Analysis of blood and urine revealed
no changes, and there was no evidence of either gross or historical pathology,
except for the eye.
Diets containing 0.005% or more diquat dichloride (1.1 mg diquat ion/kg/
day) resulted in the development of cataracts during the course of the experi-
ment. Table V-2 summarizes these results. An increasing severity of response
and a shorter latency (time until the response was observed) were observed as
the dose increased. No opacities were observed in any of tne animals at 0.0011
(0.22 my diquat ion/kg/day), the lowest dose tested..
Early cataract development was evident macroscopically by a paleness and
increased transparency of the eye and a greater clarity of blood vessels of
the iris. At this time, the authors could detect no microscopic abnormalities
of tne eye. The lens became quite opaque in later stages, and anterior and
posterior synechiae were noted. Hemorrhage into the vitreous humor and detach-
ment of tne retina were also observed. Cataract development in rats was not
influenced by the presence or absence of light or by the presence of excess
ascorbic acid in the diet. In addition, continuous exposure to diquat for a
v-16
-------
Table V-2. Effects'of Diquat on the Eye in
a 2-Year Feeding Study in Rats
Dietary
concentration
Estimated daily dose
of diquat dichloride
(nig diquat ion/kg bw)a
Effect
O.OU1
0.005
0.01
0.025
0.05
0.1
0.22
1.1
2.2
5.5
11
22
None
Slight opacities in 25?,
of test group at 12 months
Slight opacities in 25X
of test group at 12 months
Some degree of opacity in
all animals by 18 months
Opacity in some animals
at 4 months; bilateral
cataracts in all animals
by 12 months
Partial or complete
opacities in one or botn
lenses of all animals by
6 months
Assumes a final body weight of 0.4 kg and 12 g food consumption/day,
SOURCE: Adapted from Clark and Hurst "(1970).
V-17
-------
period of more than 8 weeks was necessary to produce the cataracts. It was
observed that administering a single oral dose or feeding a diet known to
produce cataracts within a few months (0.057. diquat) for a period of only 8
weeks did not lead to the formation of cataracts.
In the study with dogs (Clark and Hurst, 1970), groups of three male and
three female Alderley Park beagles, with initial body weights ranging from 3 to
12 ky, were fed diquat dichloride mixed with their food for periods of 2 to 4
years. Daily doses of either 5 or Ib mg diquat dichloride/kg bw (3.6 or 10.8
my diquat ion/kg/day, respectively) were administered for 2 years; doses of 1.7
my/kg bw (1.2 mg diquat ion/kg/day) were administered for 4 years; and doses of
0.4 or 0.8 my/kg bw (0.29 or 0.58 mg diquat ion/kg/day, respectively) were
administered for 3 years. Growth in the treated animals was comparable to that
of control animals at all dose levels. In addition, no effects on hematology,
urinalysis, blood urea, serum alkaline phosphatase, or liver function were
observed. Except for the development of cataracts, histopathological examina-
tion of organs showed no other lesions at the end of the experimental period.
Bilateral opacities of the lens in all animals occurred after 10 to 11 months
of administration of 10.8 mg diquat ion/kg/day. At a dose of 3.6 mg diquat
ion/kg/day, one lens in one animal was affected^within 11 months, and all
animals were affected in both eyes by 15 to 17 months. No effects on the eyes
were observed at 1.2 my diquat ion/kg/day for 4 years or 0.58 or 0.29 my diquat
ion/ky/day for 3 years.
Bainova and Vulcheva (1978) administered diquat in drinking water to rats
at 2 and 4 my/kg/day for 1 and 2 years, respectively. No increase in mortality
rates was noted. Histological changes were observed in lungs after treatment
with 4 my/kg/day. The minimal effective dose was 2 mg/kg/day.
V-18
-------
Makovskii (1972) administered diquat orally to rats (0.2, 2.1, and 5.3
mg/kg/day) and guinea pigs (p.l, 1.0, and 2.5 mg/kg/day) for 1 year. The
higher doses were toxic in both species. The NOAEL was 0.2 mg/kg/day for rats
and 0.1 mg/kg/day for guinea pigs. The guinea pigs appeared to be more sensi-
tive to the herbicide.
C. REPKOOUCTIVE/TERATOGENIC EFFECTS
In a dominant lethal study, diquat had no effect on fertility in male mice
administered the compound orally (Anderson et al., 1976a). Three groups of 15
male (Charles River, CD-I) mice of demonstrated fertility were dosed oral.ly
with 0.10, 1.00, or 10.00 mg diquat ion/kg, formulated in 0.5% Tween 80, for
5 days. The body weight of these animals was not specified, although age was
reported as 10 to 12 weeks. Immediately after treatment, these mice were mated
with successive groups of virgin female mice at weekly intervals for 8 weeks.
The female mice were killed 13 days after the assumed date of fertilization.
Uteri of the killed mice were assessed for live implantations and early and
late deaths. Diquat was not found to decrease fertility, as measured by preg-
nancy frequency or successful mating frequency, at any dose level. There was
no -significant increase in the number of preimplantation losses. Postimplanta-.
tion losses, as measured by (1) the number of pregnancies with one or more
early deaths, (2) the number of early deaths per pregnancy, or (3) the perxan-
tage of total implants recorded as early deaths per pregnancy, also showed no
significant increase. The authors concluded that diquat showed no induced ?ra-
or postimplantational dominant lethality'(Anderson et al., 1976a).
In a three-generation reproduction study in rats, Griffiths et al. (1965}
observed that the ingestion of diquat by the male, female, or by both parents
did not affect the fertility of tne treated animals or their offspring.
V-19
! ,
(I
-------
Ten-week-old tfi star-derived rats of unspecified body weight from the Alderlay
Park colony were fed 125 or 500 ppm diquat ion (as diquat dichloride monohydrate)
in the diet (estimated daily dose of 6.25 or 25 mg diquat ion/kg bw, assuming a
final body weight of 400 g and a daily food consumption of 20 g). Three groups
of rats (10 male, io female, or 10 of both sexes) received 500 ppm diquat (25
mg diquat ion/kg); 125 ppm diquat (6.25 my diquat ion/kg bw} was administered
to 10 male and 10 female rats.
Except for the actual mating time and the period from days 12 to 21 of
lactation, feeding continued throughout the production of three litters by each
female. The first matings occurred after the rats had received the diquat diet
for 60 days.. Representatives of the FI and Fg generations from each group
remained untreated and were mated at an age of 100 days. Treatment with diquat
did not influence the number of successful matings in the parental generation
at either dose level. The number of animals per litter reared to weaning in
each of the three reproductive periods was comparable for all groups. Thera
was no influence on growth among the progeny. Similarly, reproduction in the
Fj and Fg generations was unaffected by the treatment of the parental generation.
Diquat was not found to have significant teratogenic effects in the mousa
(Palmer et al., 1978), the rat (Griffiths et al., 1966; Moore and Wilson,
1973), or the rabbit (Palmer and Pratt, 1974) following either oral administra-
tion or treatment with diquat-containing diets. Palmer et al.. (1978) observed
tnat diquat had no significant effect on embryonic or fetal development in trie
mouse at levels of up to 4 mg diqua: ion/kg DW. Pregnant SPF albino mice (32
to 34 per dose level) obtained from :ne Alderley Park colony were used in this
study. These mice were treated wi:n daily oral doses of 0, 1.0, 2.0, or 4.0 my
diquat ion/kg bw (administered as diquat Jioromide monohydrate in aqueous
V-20
-------
solution) from day 6 tnrough day 15 of gestation. The initial body weight
range was 25 to 30 g (aye not specified). The animals were killed on day 17 of
gestation. Litter size, postimplantation Toss, and litter weight were not
significantly affected by treatment, but a reduction in mean fetal weignt (13%)
was recorded at 4.0 my/kg bw. A high incidence of minor skeletal abnormalities
was found in all diquat-treated groups. The mean- percentages of minor skeletal
abnormalities/litter were 10-.5, 20.1, 28.1, and 27.0 for the 0-, 1.0-, 2.0-,
or 4.0-mg diquat ion/kg dose groups, respectively. The incidence Of fetuses
with variant sternebrae was 1.5 to 1.6 times higher in diquat-treated groups
than in the controls. The authors concluded that diquat was not teratogenic
in mice at the 1.0-mg diquat ion/kg level. However, diquat was fetotoxic at
this dose level because it retarded ossification. Maternal toxicity (decreased
body weight gain and increase in the incidence of toxic symptoms) was also
manifested at the 1.0-my diquat ion/kg dose level.
In the reproduction study in rats by Griffiths et al. (1966), detailed
clinical and postmortem examinations were conducted on approximately half the
1,637 offspring. No skeletal or visceral abnormalities were observed, with the
exception of a single unilateral cataract in a female member of trie third
litter born to parents treated with 5UO ppm diquat (25 mg diquat ion/kg/day).
This opacity, characterized histologically as a subcapsular vacuolization of .
the lens fibers, was observed at 21 days of age.
A study by Moore and Wilson (1973) showed no evidence of cataract forma-
tion in the offspring of rats fed 5UU ppm diquat ion throughout gestation.
Groups of 20 pregnant Sprague-Dawley fats, supplied by Carworth Europe, were
fed 125 or 5UO ppm diquat ion {admii! s:ere<3 as diquat dibromide) in the diet
(estimated doses of 6.25 or 25 mg 21 ^a: ion/kg/day, assuming a food consump-
tion of 20 g/day) throughout -gestati:n. >,e initial body weight of these
V-21
-------
animals was approximately 190 g; age was not.specified. The animals *ere
either killed or allowed to deliver on day-20. No adverse effects were observed
in the number of implantations, mean number of fetuses, mean litter weight, or
sex ratio at either dose level. The total number of resorptions in treated
groups was not significantly different from that of the control group. Fetuses
delivered from animals receiving 500 ppm diquat ion had significantly lower
body weight. This effect was associated with reduced maternal food consumption
and body weight gain. A dose of 125 ppm diquat ion (6.25 mg/kg/day) had no
effect on fetal weight. Microscopic examination of the fetuses showed an
.apparent dose-related increase in subcutaneous hemorrhages in the back, nose,
jaws, forelimbs, and hindlimbs. The number of fetuses exhibiting subcutaneous
hemorrhage was 0/191, 4/238, and 12/224 in the control, 125-ppm, and 500-ppm
dose groups, respectively. The testing laboratory asserted that these lesions
were not in response to treatment with diquat. No consistent pattern of soft
tissue or skeletal anomalies was observed in the treated groups. The offspring
of four rats that were allowed to deliver after receiving 500 ppm diquat ion
{25 m^/kg/day) showed no clinical or histologica! evidence of cataract formation
at weaning. The authors concluded that diquat was without teratogenic affects
following administration in the diet of rats. A maternal and fetotoxic NQAtl
of 125 ppm (6.25 mg diquat ion/kg/day) and a teratogenic NOAEL of greater than
5UO ppm (25 mg diquat ion/kg/day) were identified in this study. >* -
Similar negative results were reported for the rabbit. Palmer and Pratt
(1974) observed that diquat administered orally to pregnant rabbits at levels
up to 5 mg diquat ion/kg bw tnrouynou: jestation had no effect on the fetuses.
Dutch rabbits (15 to 20/dose level;-, v-^lied by Cheshire Rabbit Farm Ltd.,
were orally administered 1.25, 2.5. 3- S.J ^ diquat ion/kg bw, as a solution
of diquat dibromide monohydrate in Diiije-sol OG, from day 1 through day 28 3f
V-22
-------
. Body weights on day 1 of treatment were approximately 2.2 kg; age
was not reported. Two animals that received 5 my diquat ion/kg were allowed t
deliver; the regaining animals were sacrificed on day 29. There were no sig- :
m'ficant effects on the number of implantations or resorptions or on fetal :
viability, body weight, or sex ratio. No cataracts were observed, and there ;
were no skeletal or soft-tissue abnormalities attributable to treatment. The '|
authors concluded that orally administered diquat was not teratogen.ic or cata- '
ractogenic in uterp in rabbits.
Teratogenic effects and maternal toxicity were evaluated in rats and mice ,'
administered diquat via ip or iv injections. Diquat induced abnormalities
during the prenatal development of rats (Khera et al., 1970). Single ip doses
of 7 mg diquat/kg or 14 mg diquat/kg (3.75 and 7.5 mg diquat ion/kg, respec-
tively) were administered to female Wistar rats on gestation days 6 to 11 and
13 to 15. A control group received an equal volume of vehicle (distilled
water). The females were sacrificed near term, i.e., on day 21. At the lower ;!
dose (3.75 mg diquat ion/kg/day), a high incidence of sternal abnormalities,
absence or lack of ossification of one auditory ossicle, and marked weight
reduction were observed in the embryos. At the higher dose (7.5 mg diquat
ion/kg/day), the authors reported maternal deaths or early interruption of
pregnancy and embryonic defects in surviving embryos.
Bus et al. (1975) administered, to Sprague-Dawley (CD) female rats, a
single iv dose of 15 mg diquat/kg (3 mg diquat ion/kg) on one of gestational ,1
days 7 to 21. On day 22, the numoer of live and dead fetuses plus resorbed j
fetuses was counted. An increase :n tne number of dead and resorbed fetuses
was observed. The average percentage jf jead plus resorbed fetuses was 57. ;,
The incidence of maternal death was :,"., mc':ating that a single iv doss of ii ;;
my diquat/ky produces a low level of -na^ndl toxicity. |
V-23
-------
Selypes et al. (1980) injected female mice (CFLP strain) with single ip
doses of diquat at 2.7 and 11 mg diquat/kg bw (1.4 and 5.9 mg diquat ion/kg,
respectively) on\days 9, 10, 11, and 12 of gestation. A significant increase
in the number of dead fetuses and post implantation lethality was observed at
both dose levels. There were no congenital malformations. The average fetal
weight decreased. Skeletal abnormalities included large fontanelles, wider
cerebral sutures, flat-shaped ventral nuclei of the vertebrae, and delayed
ossification in the sternum and phalanges.
D. MUTAGENICITY
Contradictory results have been reported in the literature on the mutagenic
effects of diquat. This section includes the published studies as well as a
series of unpublished assays performed to meet U.S. EPA registration requirements,
These are categorized into gene mutation assays (Category 1), chromosome aberra-
tion assays (Category 2), and studies that assess other mutagenic mechanisms
(Category 3). The findings are discussed below and summarized in Table V-3.
1. Gene Mutation Assays (Category 1)
a. Bacteria
Botn positive (Probst et al ., 1981; Wildemar and Nazar, 1982) and negative
(Moriya et al., 1983; Shirasu et al., 1976; Benigni et al., 1979; Levin et al.,
1982) findings nave been reported for reverse mutation using the Ames Salmonel la
typhimurium assay. The tests were conducted without or with metabolic activa-
tion systems (S9 from rat liver and S14 from corn). Dosages and strains used
are listed in Table V-3.
V-24
-------
Table V-3. Summary of Results From Selected Mutagenicity Studies
Test system
t
I !
Species/strain/tissue ' Dose7conc. Result Reference
Bacteria
Modified Ames test
without activation
Ames test without
activation
Ames test with and
without activation
Ames test with and
without activation
Ames test witn
activation
Ames test with rat
liver and plant
(corn) activation
Sec-assay
Liquid holding test:
forward mutation to
streptomycin resis-
tance
Spot-test: forward
mutation to galac-
tose prototrophy
Spot-test: back
mutation to
prototrophy
Salmonella typhimurium.
G46, TA1535, TA1000, C3076,
TA1537, 03052, TA1538,
TA98, and
Escherichia cgji. WP2,
WP2 uvrA-
S. typhimurium, TA100,
TA98, TA1535, TA1537 ,
TA1538, and
£. coll. WP2 her
S. typhimurium, TA1535,
TA1536, TA1537, TA1538,
0.5 to 1,000
nmol/mL
and
WP2
S. typhimurium, TA1535,
TA1537, TA98, TA100
_S. typhimurium, TA102,
TA92, TA95, TA96, TA102,
TA103, TA104, TA2638
S. typhimurium, TA100
Bacillus subtilis, H17
Rec+, M45 Rec-
E. coli
E. coli, Gal
Serratia marcascens,
a21f
Not reported
1 mg/mL
0.25, 0.5,
1.0, 2.5,
5.0, 10 ug/
plate
10 ng/plate
0.2-500 ug
act i ve
ingredient
1 mg/mL
Not reported
Not reported
Not reported
Probst et al
(1981) (
Moriya etj al
(1933) '.
Shi rasu >t a
(1976) ;|
ni sit a
(1979^
Levin eti'al .
(1982) ',
j^iIda-nan ;artd
Nazsr (1^32)
Shirasu tit a
(1976) ',
Fahrig (i!97d
Fahrig (;L97.
-------
Table V-3. (continued)
Test system
Species/strain/tissue
Dose/cone. Result Reference
Bacteria (continued)
Repair test
8-Azaguanine (AG)
resistance test
Yeast/fungi
Mltotic gene
conversion
Liquid holding
test: mitotic
jene conversion
Lethal recessive
damage
8-AG resistance
and methionine
suppression
Mammalian cells
Scheduled DNA
synthesis
Scheduled DNA
synthesis
Unscheduled DNA
synthesis
Unscheduled DNA
synthesis
Unscheduled DNA
synthesis
. Unscheduled DNA
synthesis
S. typhimurium. TA1538,
7A197S
^. typhimurium. his G46,
TA92, TA1535; TA92, TA100
S, cerevisiae
A. nidulans, 35
Rat thymocytes
Human lymphocytes
Rat hepatocytes
Human epithelial-1ike
cells
Human lymphocytes
Human fibroblast cells,
VA-4
1U ug/plate
0.1, 0.25, .
0.5, 1,0 ug/
plate
Sacchargmyces cerevislae, 1 ug/mL
^4
Not reported
Asperjillus nidulans. P3 10 my/mL
10 ntg/inL
100, 500,
1,000 ug/mL
500 ug/mL
0.5 to 1,000
nmol/roL
20, 100, 1,000, *
2,000 ug/mL
500 ug/mL +
1, 10, 100,
1,000 urt
Senigni et al
(1979)
Benigni ec al
(1979);
Biynami and
Crebelli (19T
Siebert and
Lemperle (197
Fahrig (1974)
Senigni et a1,
(1979)
Senijni et al
(1979)
Rocchi et al .
(1980)
Rocchi et al.
(1980)
Proost at al .
(1931)
Beniyni. et al
(1979)
Rocchi st al .
(1980)
Ahned et al .
V-25
-------
Table V-3. (continued)
Test system
Species/straih/tissue
Dose/cone. Result Reference
Drosophlla
Muller-5 test:
lethal recessive
damage
House
Dominant lethal
assay
Drosopni 1 a tnelanogaster 0.2 ug/ml
Mouse, Swiss-Webster
76 mmol/kg,
Pasi at a|j.
(1974) i;
V-27
-------
Negative results were also reported in the forward mutation (to strepto-
mycin resistance and galactose prototrophy) assays with Escherichia coli.
However, positive results have been reported in S,. typhimurium utilizing the
8-azaguanine (8-AG) resistance system.
b. Eucaryotic microorgani sms
Oiquat was positive in Aspergillus nidulans in the methionine suppression
and 8-AG resistance system (Benigni et al., 1979). It also induced lethal
recessive damage in A. nidulans.
c. Sex-linked recessive lethal (SLRL) mutations in Drosophila melanogaster
Diquat failed to induce SLRL mutations when evaluated in £. melanogaster
usiny the Muller-5 genetic test, which detects recessive lethality in the
r
X-chromosome (Benes and Sram, 1969). Oiquat was injected into the abdomen at 2
uL/fly (the highest possible concentration, slightly under the lethal or sterile
threshold).
2. Chromosome Aberration Assays (Category 2)
Pasi et al. (1979) were unable to induce dominant lethal mutations in mica
injected intraperitoneal ly with 76 mmol/kg.
3. Other Genotoxic Effects (Category 3)
a. Differential toxicity in bacteria
Diquat was not mutagenic in rec-assay with Bacillus sufatilis, which meas-
ures the growth inhibition zones for H17 Rec+ and M45 Rec- cells (Shirasu et
al., 1976).
V-23
-------
b. Mitotic gene conversion
Siebert and Lemperle (1974) tested diquat for its induction of mitotic !;
gene conversion in a diploid strain of ascomycete Saccharomyces cerevisiae -
heteroallelic at 2 loci. Diquat showed weak genetic activity. Fahrig (1974), ;
on the other hand, reported negative results in mitotic gene conversion at ade2 ':
and trp5 loci of S^. cerevisiae. .
c. Scheduled and unscheduled DNA synthesis (UPS) ,;
Of six studies on the effect of diquat on .scheduled and unscheduled DNA
synthesis in human cells, five reported positive results (Benigni et al., 1979;
.1
Rocchi et al., 1980; Ahmed et al., 1977). One negative finding was reported
with rat hepatocytes (Probst et al., 1981). Diquat inhibited scheduled ONA
synthesis in rat thymocytes and human lymphocytes to the same degree (Socchi et
al., 1980). This inhibition was comparable to that obtained with unscheduled
DNA synthesis of human lymphocytes. Ahmed et al. (1977) studied the effect of
diquat on the induction of unscheduled DNA synthesis in SV-40 transformed nunan
cells (VA-4). An increase in unscheduled ONA synthesis was observed in cell
cultures treated with diquat without metabolic activation. Metabolic activa-
tion did not significantly alter the activity of diquat.
4. Miscellaneous
Benigni et al. (1979) assayed diquat (see Table V-3) in a number of muta-
genicity test systems (Ames assay, resistance to 8-azaguanine, and repair :es:
in Su typhimuri'um; gene mutations and lethal recessive damage in £. nidulans;
UOS in human epithelial-1ike cells). They concluded that diquat was able to
induce gene mutations, both in prokaryotic (S^ typhimurium) or eukaryotic
*
microorganisms (£. nidulans), only in forward-mutation systems. The use of
V-29
-------
repair-proficient strains of S.. typhimurium enhanced this activity. From this
result and the negative response obtained in the Ames test, the authors
concluded that diquat may be able to induce damage to the gene level (small
deletions, cross-links, strand breaks), but not gene mutations of the
base-substitution or frame-shift type. In addition, the authors concluded that
diquat may cause premutational lesions. This damage may be repaired, resulting
in gene mutations, or my remain unrepaired, resulting in cell death.
E. CARCINOGEN I CITY
Data from four chronic toxicity studies with rats and one with mice suggest
that diquat lacks carcinogenic potential. Two-year feeding studies in which
rats were administered 15, 25, or 75 mg diquat ion/kg food (approximately 0.75,
1.25, or 3.75 mg/kg/day, respectively) (Royerson and Broad, 1978) and dose
levels of up to 0.1* (estimated dose of 36'mg diquat ion/kg/day) in the studies
of Clark and Hurst (1970) failed to show evidence of induced tumors. Sainova
and Vulcheva (1978) also observed no evidence of malignancy in a 2-year feeding
study. Male and female Wistar rats (50 per dose level, 40 controls, initial
body weight 120 to 140 g, age not specified) were administered doses of diquat
dibrcmide in their drinking water at a concentration of 2.0 mg diquat/kg/day
for 1 or 2 years or 4.0 mg diquat/kg/day for 2 years. Histological examination
revealed no marked aberrations in liver, kidney, or myocardium in treated
animals. However, lungs from rats in the high-dose group were swollen and
exhibited desquamation of epithelial cells, thickening of the alveolar parti-
tions, and hyperplasia of peri bronchial lymph tissue. No signs of malignancy
were found.
Colley et al . (1985) evaluated :ne carcinogenic .potential of diquat di&ro-
mide in a 104-week dietary study in Charles 3iver CO rats. The experimental
V-3U
-------
details of this study are reported under chronic toxicity. The conclusion of ti|e
authors was'that administration of this herbicide in the diet at levels up to
-.'
375 pprn (17.16 mg diquat ion/kg bw/day) had no carcinogenic effects. At '!
if
present, however, the data which support this conclusion are insufficient sinceM
!
historical control data for relevant target tissues as well as statistcal I'
if
analyses of both neoplastic and non-neoplastic lesions were not submitted with ij
'i
the report. . :'
Ben-Dyke et al . (1975) studied the effect of diquat on tumor incidence in
CD-I mice (4 weeks old, initially). Diquat (as the dibromide monohydrate) was !
given in the diet at doses of 30, 150, or 300 to 500 mg diquat ion/kg food
(estimated daily doses of 4.5, 22.5, or 45 to 75 mg diquat .ion/kg bw, respec-
tively, assuming a final body weight of 20 g and a daily food consumption of 3 jj
g). A total of 60 male and 60 female mice were used for each dose. After 80
weeks, no treatment-related incidence of neoplasms was observed-, however,
the report was inadequate because too much of the important histopathological
data were missing and there were ambiquities in the submitted data..
F. SUMMAKY
Diquat administered by the oral route exhibits moderately acute toxicity
in mammals. Oral LDsu values for various species ranged from 26 to 430 mg ^.^^
diquat ion/kg bw. The potency of diquat when administered by subcutaneous
injection may be up to 20 times greater than when administered by the oral
route. It has been suggested that this is due to the poor absorption of diquai
from the GI tract. Rats receiving an LDso dose (166 mg diquat ion/kg bw)
exhibited lethargy, signs of piloerec:ion, and weight loss, and they excreted
feces of a mucoid, ropey form with a cnaracteristic greenish color. Gross
abdominal swelling, muscular twitching, and an erratic gait were also observed.
V-31
-------
The most notable effects of acute oral doses were an increase in gastrointesti-
nal water content and hemoconcentration. Diquat has a profound effect on body
water distribution; dehydration may play a key role in mortality.
Diquat has an acute physiological effect on the GI tract. Oral doses pro-
duce a massive redistribution of body fluids characterized by an increase in
the water content of the GI tract. In rats, the LOAEL for an increase in water
content of the lumen of the GI tract in 24 hours was an oral dose of 18.4 my
diquat ion/kg bw, the lowest dose tested. This physiological change was accom-
panied by minimal histopathology in rats. Exfoliation of the gastrointestinal
tract epithelium was observed in monkeys that died after receiving single oral
doses of 100 to 400 mg diquat ion/kg bw. Perforation of the stomach wall was
noted at LDjo doses in dogs (100 to 200 mg ion/kg bw) and rabbits (100 mg
ion/kg bw, respectively).
v
A marked decrease in renal excretory function was also observed in rats
as a result of a single oral dose of 99 mg diquat ion/kg bw. At 166 mg diquat
ion/kg bw, hemoconcentration and a significant reduction in renal plasma flow
were observed. Minimal pathological changes in the kidney were observed in
rats at LDgQ dose levels. Therefore, researchers have concluded that tne
effects on renal function are primarily a result of body fluid redistribution.
Pathological changes were, however, observed in kidneys of monkeys receiving
single oral doses of 100 to 400 mg diquat ion/kg bw.
Effects on the liver were minimal in rats receiving acute lethal doses of
diquat intraperitoneally. An increase in liver glycogen and blood glucose
appeared to be mediated by altered adrenal secretion. Selenium-deficient rats,
given diquat (3.6 mg/kg) intraperi coneiM/, exhibited rapid and massive liver
V-32
-------
necrosis accompanied by a marked increase in hepatic lipid peroxidation.
"Hepatic effects in monkeys were minimal after oral doses of diquat.
Oral administration of diquat for 4-1/2 months (2.1 and 4.3 my/kg/bw/day)
to rats, produced lung damage characterized by papillomatous proliferations of
the bronchial and bronchiolarepithelia. The changes appeared to be dose-related!.
Moderate to severe alveolar damage has been reported following intratracheal or j
intraperitoneal administration of diquat in mice. t
No signs of irritation to the digestive mucosa were observed when diquat
was administered in drinking water to rats (500 and 1,000 mg/L for 20 and 8 daysjj
respectively) and rabbits (100 and 500 mg/L for 6 and 10 days, respectively).
In a 4-week dietary feeding study with rats, reduced weight gain, reduced food <[
!t
intake, and liver involvement were noted at dose levels ranging between 17 and !
39.7 mg diquat ion/kg/day.
I
Chronic feeding studies were conducted in rats, guinea pigs, and dogs. ;
In a 1- to 2-year study in rats, the minimal effective dose of diquat was 2
mg/kg/day in drinking water. In a 2-year study with rats, cataractogenic
effects were observed in animals treated at higher dose levels (3.28 and 17,16 ;>
mg diquat ion/kg/day). A NOAEL of 0.22 mg diquat ion/kg/day was identified.
In another 2-year study with rats, food consumption and growth rate were
reduced at the highest dose tested (about 36 mg diquat ion/kg/day). Hematologi-'!
cal examination, urinalysis,. and gross and microscopic pathological examination
showed no effects (with the exception of the eye) at any treatment level tested.
A diet containing 0.005% diquat en chloride (approximately 1.8 mg diquat ion/'
-------
effect was observed in rats fed a diet of 0.0(J1% diquat dichloride (0.36 mg
diquat ion/kg/day}. In dogs, no effects on the eyes were observed at a concen-
tration of 1.2 mg diquat ion/kg/day for * years or at a dose level of 0.58 mg
diquat ion/kg/day for 3 years. The LOAEL for dogs was 3.6 mg diquat ion/kg/day.
Guinea pigs were found to be more sensitive to diquat (NOAEL a 0.1 mg diquat
1 on/kg/day) than rats and dogs.
The results on the mutagenicity of diquat reported in the literature are
contradictory. Both positive and negative findings have been reported in the
Salmonella assay, unscheduled DNA synthesis, and mitotic gene conversion assay.
While diquat induced recessive lethal damage in Aspergillus, it failed to do
so in Drosophila.
Diquat did not cause infertility in male mice receiving five daily oral
doses of 10 mg diquat ion/kg bw. In a three-generation study, rats fed levels
of up to 5QO ppm diquat (estimated daily dose of 25 mg diquat ion/kg bw) showed
no effect on the reproduction of the parental, f\, or ?% generations. Signifi-
cant teratogenic effects were not observed following oral administration of
diquat in the mouse, rat, or rabbit. However, teratogenic effects were observed
when diquat was administered to rats or mice via ip or iv injections. An
increase in the number of dead and resorbed fetuses was observed in rats admin-
istered a single iv dose of 8 mg diquat ion/kg. Skeletal abnormalities wer>*-'
found in mouse and rat embryos after treatment of darns with single ip doses of
diquat (1.4 and 5.9 mg diquat ion/kg for mice and 7.5 my diquat ion/kg for
rats} during gestation.
Four feeding studies in rats and one in mice, in which daily doses of up
to about 75 mg diquat ion/kg bw wera yiven for periods up to 2 years, failed
to demonstrate tumor induction. However, the data from two of these studies
were not sufficient.
V-34
-------
-------
VI. HEALTH EFFECTS IN HUMANS
A. CLINICAL CASE STUDIES
A number of cases of diquat poisoning have been reported in the literature.
Host of these cases involved accidental poisoning or suicide; consequently, it
is difficult to determine exact levels ingested. Table VI-1 summarizes the
available information on nonoccupational exposure to diquat. In 6. of 10 cases
in which diquat was ingested, orally, a fatal outcome occurred 1 to 7 days after
ingestion.
A case of accidental diquat poisoning in a 2-1/2-year-old boy was des-
cribed by Powell et al. (1983). The child (weight 13 kg) ingested an unknown
amount of 2U% diquat solution left in a soft-drink bottle. Vomiting was induced
by pharyngeal stimulation upon discovery. The child was moved to an emergency
room 3 hours after ingestion. His stomacn was lavaged with water containing
Fuller's earth. Hemoperfusion was performed six times in an effort to lower
the body diquat burden. However, the child died 143 hours after ingestion.
Progressive neurologic dysfunctions were observed preceding his death. Post-
mortem examination revealed brain stem infarction, purpura, and multiple muco-
sal ulcers of the mouth. The lungs displayed nonspecific bronchopneumonia, and
the kidneys were pale and edematous. The liver appeared to be normal.
Vanholder et al. (1981) described a case involving a 16-year-old girl who
intentionally ingested about 50 mL of Reylone (20U g diquat ion/L in aqueous
solution). Almost immediately afts*- swallowing the herbicide, the girl exper-
ienced abdominal cramps, vomiting,
-------
Table VI-1. Sumnary of
Case Studies of Oiquat
Reference
Oreopoulous and
McEvoy (1969)
Wei rich (1969)
Schonborn et al .
(1971)
Okonek and Hoffman
(1975)
Pel et al. (1976)
Vannolder et al .
(1981)
Van holder et al .
(1981)
Powel 1 et al .
(1983)
McCarthy and Speth
(1983)
Mahieu et al .
(1984)
Wood et al . (1976)
Williams et al . (1986)
Age (yr),
sex
18, M
43, M
25, M
43, F
53, M
16, F
60, F
2-1/2, M
23, M
33, M
45, M
24, M
II
!l
Approximate quantity
Exposure ingested ml
route (g diquat ion)3 Outcome j|
Oral
Inhalation,
oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Inhalation
Inhalation
5 (l)l>
Unknown
15 (3)b
Unknown
5 (3)b
- 50(10)
20 (4)
Unknown
300 (60)
300 (60)
5 (3)b
Unknown
!)
ij
Survival!!
't
Survival'
Death :;
(7 days))
» j 1 1|
DeaM \
(2 days) .
Survival ':
Death j|
(1 day) i]
|i
Oeatn ;|
(5 days) jj
n
Death t.
(143 hr) ;;
Death
(1 day) :
Survival
Survival
Survival .
1
' "" '" f°ulations ar* 200'g/L diquat ion in aqueous solution *
^Estimated by Vannolder et al. (1981). !|
VI-2
-------
exposure, the patient became deeply comatose and was transferred to the inten-
sive care unit of another hospital. Her abdomen became distended, and she
experienced cardiorespiratory arrest soon after admission. The patient died 24
hours after an intake of about 10 g of the diquation.
Another case of diquat poisoning has been described by Oreopoulous and
McEvoy (1969). An 18-year-old male accidentally ingested a mouthful of undi-
luted Reglone (200 diquat g/L in aqueous solution) that was contained in a
soft-drink bottle. The subject reportedly spat most of it out, but stated
that he was certain that he swallowed some small quantity (approximately 5 ml,
or 1 g diquat). He felt progressively worse and developed diarrhea after about
10 hours. By 56 hours after ingestion, he had developed an ulcer in his throat
and had difficulty swallowing. He showed no signs of abdominal distension.
Despite this patient's lack of symptoms, he was treated by forced diuresis.
This treatment was based on clinical reports of incidents of paraquat poisoning
in which patients had experienced progressive renal failure. A good diuretic
response was obtained in the diquat-poisoned man, with only slight changes in
his blood electrolyte levels. He continued to excrete diquat in his urine
until 9 days after ingestion; diuresis was prolonged for 2 additional days.
In this case, the patient was released from the hospital on the 22nd day, witrs
no apparent kidney damage.
In general, Vanholder et al . (1981) reported that acute poisoning of
humans with doses of Reglone over 20 ml (>4 y diquat) resulted in necrosis of
the heart, liver, and kidneys, and led to cerebral hemorrhage. Death usually
resulted within several hours to a few days. Ingestion of smaller doses of
Reglone (<5 ml or 1 g diquat) usually resulted in vomiting, diarrhea, and
damage to the GI tract and kidneys. Acjte renal failure, vascular collapse,
VI-3
-------
and decreased cardiac output are common complications. Unlike paraquat poison-
ing, where pulmonary fibrosis is the most life-threatening complication, indivi
duals who have survived diquat ingestion do not appear to suffer permanent
pulmonary damage if they receive prompt treatment. Generally, this treatment jj
involves (1) immediate gastric lavage, with Fuller's earth (a clay substance !|
that adsorbs diquat), to remove as much of the compound as possible and to ;,
reduce the local and system'c toxicity; and (2) hemoperfusion to increase the
rate of elimination of diquat from the blood.
i
8. EPIDEMIOLOGICAL STUDIES
No epidemiological studies of the health effects of diquat have been
reported in the literature (WHO, undated). Observations of agricultural
workers, who handle diquat as a crystalline powder or are exposed to it through!)
^
accidental inhalation during spray application, reveal nail growth disturbances'1
and irritation of the upper respiratory tract. According to Clark and Hurst
(1970), if a 20% diquat solution comes into contact with the nail base, nail
growth is disturbed. Shedding of the nail has been noted with prolonged expo- ,
sure; however, regrowth of the nail occurs once exposure is discontinued. More!
quantitative data are lacking. Clark and Hurst (1970) also reported inflamma-
tion and bleeding of the nasal mucosa in workers who handled diquat powder in
tne field or the laboratory.
Cataracts have not been reported in humans following diquat exposure (FAO/i;
WHO, 1978). I
I;
C. HIGH-RISK SUBPOPULATIONS ;
t
Wojeck et al . (1983) monitored :ie eoosure of workers who applied diquat ;
(diquat dibromide, 35.3% active i-^reaien:} from airboats to a southwest Florida
VI-4
-------
waterway containing water hyacinths and hydrilla. Dermal alpha-cellulose pads
were attached at 10 locations on the body to measure dermal exposure. Wilson
"Dustite" respirators with filter pads were used to measure respiratory expo-
sure. The dlquat applicators received minimal exposure. Respiratory exposure
was less than 0.1* of the total body exposure. Urine samples were negative.
The authors concluded that diquat poses "little possible acute danger to appli-
cators, mixers, or drivers."
0. SUMMARY
A few cases of known diquat exposure have been reported in the literature.
Six of the people described in these cases, all of whom swallowed more than
about 15 ml Reg lone (3 g. diquat), died of complications involving the GI tract.,
brain, and kidneys. Ingestion of 5 ml Reglone (1 g diquat) does not appear to
be lethal, but patients exhibited symptoms of GI tract-and renal damage.
Treatment for diquat poisoning involves the prompt use of gastric lavaye to
reduce tne absorbed dose and therefore prevent clinical sequelae.
VI-5
-------
VII. MECHANISMS OF TOXICITY
The mechanism of diquat toxicity is not clear. In plants, light-induced ;
formation of the diquat free radical appears to be an essential step in phyto-;!
toxicity (Conning et al., 1969). Most proposals' regarding diquat toxicity in i
animal cells have also centered around free radical formation.
A. FORMATION OF FREE RADICALS
Gage (196*3) reported that rat liver microsomes, incubated in. the presence)
of NADPH, could reduce diquat di-cations (18 to 180 mg/L) to the mono-cation :|
free radical. This process is not inhibited by carbon monoxide. In the absence
i
of Og, the monocation accumulates, but admission of 02 results in reoxidation ji
of the radical (reforming the original di cation) with concomitant formation o
superoxide and hydrogen peroxide (Pirie et al., 1970). Thus, diquat stimulate!;
Og consumption by rat liver microsomes. In the eye, diquat free radicals may
be formed via a light-dependent mechanism, provided a suitable electron donor (|
i
(e.g., amino acids or other constituents of ocular fluid) is present (Pirie et |
al., 197U). The component- of the microsomal fraction mediating NADPH -dependent
ji
reduction of diquat to the free radical is usually assumed to be the mixed- ;;
function oxidase system. This is supported by the observation of Krieger et ;i
al . (1973) that diquat is an inhibitor of aldrin epoxidation in rat liver
microsomes (half-maximal inhibition at 1.2 mg/L). :i
i
The relationship between free radical formation and cell injury is not ij
clear. Baldwin et al . (1975) compared the ability of microsomes prepared froinji
rat lung, kidney, and liver tissue to form free radicals from diquat (17 mg/L)s!
Liver microsomes were most effective in forming radicals, with Tuny microsomesjj
intermediate and kidney microsomes lease effective. As noted earlier, diquat
VII-1
-------
causes more tissue Injury in the kidney than in the liver (see Section VI.8).
Moreover, in lung microsomes, diquat led to more radical formation than did
paraquat. Since paraquat has been found to be more toxic to rat lung than
diquat, the authors concluded that no obvious relationSMp exists between the
extent of radical formation from these compounds and their ability to damaye
cells.
8. LIPID PEROXIDATION AND ROLE OF OXYGEN
Free radical formation often leads to peroxidation of cellular lipids, and
this has frequently been proposed as a mechanism of radical-induced cell injury
(Conning et al., 1969). Carmines et a!. (1981) demonstrated that diquat (3.52
mg 1on/L) inhibits growth of cultured P388Di "macrophage-like" cells without
causing measurable Hpid peroxidation. Talcott et al. (1979) reported that
diquat 1s 10 to 15 times more potent than paraquat in causing lipid peroxida-
tion in mouse lung microsomes (half-maximal concentrations » 1.1 versus 14.7 tng
ion/L, respectively), even though diquat is much less toxic to the lung than is
paraquat. This observation suggests that lipid peroxidation may not be tightly
i
linked to diquat toxicity.
Under aerobic conditions, superoxide and-hydrogen peroxide are also gen-
erated as a consequence of free radical formation. These oxides are frequently
considered likely mediators of cell injury. Talcott et al. (1979) showed that
diquat stimulated superoxide and hydrogen peroxide formation in mouse liver
microsomes at 2 and 10 times their respective endogenous rates. However, super-
oxide was formed at much lower doses of diquat (50% effect at 0.1 mg diquat
ion/L) than were required to produce lipid peroxidation (50% effect at 2.6
mg/L). Moreover, addition of superoxide di smutase or catalase did not prevent
VII-2
-------
lipid peroxidation. Thus, the authors concluded that generation of superoxide
or hydrogen peroxide was not closely associated with lipid peroxidation.
In a study by Younes et al. (1985), oral doses of diquat (100 mg/kg bw) !
failed to elicit in vivo lipid peroxidation (as evidenced by ethane exhalation) }i
in 18 male NMRI mice. However, enhanced ethane production resulted when ani-
mals were intraperitoneally administered Fe2+ (20 mg/kg) 30 minutes before '
diquat administration. Additional pretreatment-with phorone, which stimulates >
glutathione (GSH) depletion, further enhanced ethane production in rats. The
authors concluded that redox cycling compounds such as diquat do not initiate
lipid peroxidation by themselves, but can stimulate the iron-induced system.
Oxidant stress has been cited as a possible factor in diquat-induced
toxicity. A 13-fold increase in plasma glutathione disulfide levels was
observed by Adams et al. (1984) i hour after male Sprague-Oawley rats (250 to
350 g) received a single ip dose of 0.12 mmol diquat/kg bw. Plasma GSH concen- i
trations did not change significantly during the experiment. A sharp rise in j
il
plasma glutathione disulfide concentration, in the absence of an increase in i!
j :
plasma GSH, suggested to the authors that diquat undergoes a redox cycling with ;
the generation of reactive oxygen intermediates, a process that may be relevant
to the chemical's mode of action.
i
Smith et al. (1985). supported this hypothesis when they reported that'the
severe oxldant stress induced by diquat and reflected by extensive biliary
efflux of oxidized glutathione (GSSG) may be the most probable source of acute
lethal hepatic injury in animals. The investigators studied Sprague-Oawley
rats, which are resistant to diquat-related hepatic necrosis, and Fischer rats,
which exhibit hepatotoxicity following diquat administration. Eighty-seven
rats each received a single ip injection of saline, 0.05, 0.10, 0.2U, or 0.3U !i
VII-3
-------
mmol diquat/kg bw. Dramatic increases in plasma trans aminases were observed
In the Fischer rats within 3 hours of the 0.10- and 0.20-mmol/kg injections,
indicating a rapid onset of damage. Hepatic enzyme activities in these animals
were approximately 20 to 40 times greater than control values; activities were
proportionately higher at 6 hours for the 0.20-mmol/kg group. Only slight
increases in plasma transaminases were observed in the Sprague-Dawley rats that
showed minimal hepatic damage.
At the 0.10-mmol/kg dose, the excretion rate of GSSG in Fischer rats was
four times that of Sprague-Dawley rats. Other parameters, i.e., hepatic gluta-
thione peroxidase and reductase activities, hepatic ascorbic acid content,
NADPH, and protein sulfhydryls, were similar in both strains and remained
virtually constant throughout the experiment. The lipid hydroxy acid content
of the hepatic lipids diquat-treated animals was approximately twice that of
saline-treated controls, with only small increases in 11-, 12-, and 15-hydrox/-
eicosatetraenoic acids, reflecting diquat-induced peroxidation of hepatic
lipids. These findings indicated to the authors (Smith et al., 1985) that
generalized destruction of membrane lipids by auto-oxidation did not appear to
be a feature of diquat hepatotoxicity. Finally, a 50% drop in nonprotein sul.-
fhydryl (NPSH) levels, which was restored to normal within 6 hours, was believed
to result from GSSG efflux and also appeared unrelated to hepatic necrosis.
GSSG concentrations remained normal, while levels of NPSH and glutathione
(GSH) increased in the lungs of Wistar-derived, Alderley Park male rats (190 to
230 g) at 2, 8, and 24 hours postadministration of a single subcutaneous dose
of diquat ion (20 mg/kg bw) (Keeliny and Smith, 1982). This early and persis-
tent biochemical effect of diquat is not fully understood, but the authors
suggested that the herbicide may interfere with regulating normal redox status
VII-4
-------
In the lungs. However, oxidation of NADPH and GSH, which was coincidental with
lung tissue damage in paraquat-treated rats, did not occur in diquat-dosed
animals. The authors commented that although diquat causes redox stress in the"
lungs, the chemical's effects may be generally distributed among lung cell
types and not specifically targeted (as with paraquat) for alveolar epithelial
cells.
C. SUMMARY
The chemical mechanism of diquat toxicity is not clear. Lipid peroxida-
tion has been implicated in diquat-induced tissue injury, but destruction of '!
membrane lipids by auto-oxidation has not consistently been found. The genera-
tion of oxygen-reactive species that accompanies diquat metabolism may play a .
role in the chemical's mode of action, but the exact method by which these
compounds act in association with diquat has not yet been elucidated. Finally,1
although most investigators consider that formation of the diquat free radical jl
(either by metabolic reduction or by a nonenzymic photochemical reaction) is an''
essential step in toxicity, the biochemical pathways linking radical formation
to cell injury have not yet been resolved.
1
VII-5
-------
VIII. .QUANTIFICATION OF TOXICOLOGICAL EFFECTS
The quantification of toxicological effects of a chemical consists of an
assessment of noncarcinogenic and carcinogenic effects. Chemicals that do not
produce carcinogenic effects are believed to have a threshold dose below which
no adverse, noncarci nogenic health effects occur, whereas carcinogens are
assumed to act without a threshold.
A. PROCEDURES FOR QUANTIFICATION OF TOXICOLOGICAL EFFECTS
1. Noncarcinogenic Effects
In the quantification of noncarcinogenic effects, a Reference Dose (RfD),
formerly called the Acceptable Daily Intake (ADI), is calculated. The RfD is
an estimate of a daily exposure of the human population that is likely to be
without appreciable risk of deleterious health effects, even if exposure occurs
over 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 (UF). The
RfD is calculated as follows:
- RfD = (NOAEL or LOAEL) 3 mg/kg bw/day
Uncertainty factor
Selection of the uncertainty factor to be employed in the calculation^
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 fie guidelines proposed by the National
Academy of Sciences (NAS, 1977, I9ii as follows:
VIII-1
-------
o An uncertainty factor of 10 is generally used when good chronic or
subchronic human exposure data identifying a NOAEL are available and
are supported by good chronic or subcnronic toxicity data in other
species.
o 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 yood chronic or
subchronic toxicity data identifying a LOAEL in humans are availaole.
o 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-
possible intra- and interspecies differences. Additional considerations not ,!
incorporated in the NAS/OOW guidelines for selection of an uncertainty factor '
include the use of a less-than-li fetime study for deriving an RfD, the signifi- j{
cance of the adverse health effect, and the counterbalancing of beneficial
effects. !;
From tne RfO, a Drinking Water Equivalent Level {DWEL) can be calculated. ;,
The DWEL represents a medium-specific (i.e., drinking water) lifetime exposure r
at which adverse, noncarci noyenic nealtn effects are not expected to occur. ',
The DWEL assumes 100% exposure fron j-:-umy water. The DWEL provides the non- |
caryinogenic health effects basis ":>- -s:aoi i shiny a drinking water standard.
From ingestion data, the DWEL is cter-.ec is Allows:
VI11-2
-------
RfO x (body weight in kg) , m /L ( /L)
Drinking water volume in L/day
where:
Body weight = assumed to be 7U kg for an adult.
Drinking water volume » assumed to be 2 L per day for an adult.
In addition to the RfD and the DUEL, Health Advisories (HAs) for exposures
of shorter duration (One-day, Ten-day', and Longer-term HAs) are determined.
The HA values are used as informal guidance to municipalities and other organi-
zations when emergency spills or contamination situations occur. The HAs are
calculated using a similar equation to the RfD and DWEL; however, the NQAELs
or LOAELs are identified from acute or subchronic studies. The HAs are derived
as follows:
HA = (NOAEL or LOAEL) x (bw) = mg/L ( ug/L)
( L/day) x (UF)
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 tnan 30 days' duration. A Longer-
term HA is derived for both a lU-kg cnild and a 7U-kg adult and assumes an
VIII-3
-------
exposure period of approximately 7 years (or 10% of an individual's lifetime).
A Lonyer-term HA is generally derived from a study of subchronic duration j
(exposure for 10% of an animal's lifetime).
2. Carcinogenic Effects
The EPA categorizes the carcinogenic potential of a chemical, based on
the overall weight of evidence, according to the following scheme:
o Group A: Known Human Carcinogen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
o Group B: Probable Human Carcinogen. Sufficient evidence of carcino-
genicity in animals with limited (Group Bl) or inadequate
(Group 82) evidence in humans.
o Group C: Possible Human Carcinogen. Limited evidence of carcinoyeni-
city in animals in the absence of human data.
4
o Group D: Not Classified as to Human Carcinogenicity. Inadequate human
and animal evidence of carcinogenicity or for which no data
are available.
o Group E: Evidence of Noncarcinogenicity 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 :o t?e classification of the contaminant
as a known, probable, or possible fiuian car;mogen, mathematical models are
tfIII-4
I
<»
-------
used to calculate the estimate of excess cancer risk associated witn 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 hunan
doses. This conversion includes correction for noncontinuous exposure, less-
than-lifetirae studies, and for differences in size. The factor that compen-
sates for the size difference is the cube root of the ratio of the 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 cor-
related 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, Ueibull, logit,
and probit. There is little basis in the current understanding of the biologi-
cal 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 jncertainty due to the systematic and
random errors in scientific measure-lent. In most cases, only studies usiny
VIU-5
-------
Table VIII-1. Summary of Candidate Studies for Derivation
of the One-day Health Advisory" for Oiquat
Reference
Species Route
Exposure
duration
Endpoints
NOAEL
(my diquat
ion/kg/day)
LOAEL
(mg diquat
ion/!
-------
laooratory animals nave been performed. Thus, there is uncertainty when tne
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 laboratory 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
laboratory animals or humans. Dose-response data usually are available only
for hiyn 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 contami-
nant, additional uncertainty results from a lack of information about possible
synergistic or antagonistic effects.
B. QUANTIFICATION OF NONCARCINOGENIC EFFECTS FOR DIQUAT
Table VIII-l summarizes the studies considered for calculation of the
One-day HA value for diquat. The acute oral toxicity study in rats by Chevron
Chemical Company (1981} was not selected for calculation of One-day HA because
the LUAEL in this study (358 mg diquat ion/kg) is much higher than oral LOsos
reported by Clark and Hurst (1970), Gaines and Lindler (1986), Plant Protection
Ltd. (1960), and ICI (1962, 1982) (125, 121 to 147, 214 to 237, and 215 to 235
mg diquat ion/kg, respectively).
The study by Crabtree et al . (1977) has been selected to serve as tne
basis for the One-day HA for a 10-kg child. In this study, multiple single
oral doses of diquat (0, 18.4, 64.5, 111, 166, or 221 mg diquat ion/kg bw) were
employed and a LUAEL (18.4 mg diquat ion/kg bw) for hemoconcentration and f'ui.a
accumulation in the GI tract of rats was identified. A weakness of this stjdy
was that a NOAEL could not be identified; tne LOAEL of 18.4 mg diquat ion/
-------
Assumptions regarding absorption are not required in the One-day HA calcu-
lations because the LOAEL is expressed in terms of an intake dose rather than
an absorbed dose.
1. One-day Health Advisory
The One-day HA for a 10-kg child is calculated as follows:
(18.4 mg/kg/day) (10 kg) a 0.18 rag.diquat ion/I (rounded to 2UO ug/L)
(1 L/day) (l.OUO) y
where:
18.4 mg/kg/day « LOAEL for hemoconcentration and an increase in water '
. content of the GI tract following acute oral exposure in ,
rats (Crabtree et al., 1977).
10 kg assumed weight of a child. ;
1 L/day « assumed water consumption of a 10-kg child.
i
1,000 « uncertainty factor, chosen in accordance with NAS/ODW
guidelines for use with a LOAEL from an animal study. ||
2. Ten-day Health Advisory .!
No adequate data were found in the available literature for calculating
the Ten-day HA value for diquat. Consequently, the Longer-term HA value (calcu-
lated below) will be taken as a conservative estimate of the appropriate Ten-
day HA value.
The study by Chevron Chemical Company (1981) was considered but not
' . i
selected to serve as the basis for the Ten-day HA values for a 10-kg child. i,
I:
Rats were administered diquat orally for 4 weeks at 6.7, 17, 29.6, and 39.7 my
diquat ion/kg/day. Animals in all test groups, except the 6.7-mg diquat ion ,!
test group, exhibited reduced body weight gain, reduced food intake, low SG?T,
VIll-8
-------
and nigh serum cholesterol levels. No histopathological changes were observed
in any group. Therefore, 6.7 mg diquat ipn/kg/day appears to be the NOAEL in
rats. The Ten-day HA for a lU-kg child, based on a NOAEL of 6.7 mg/kg/day, and
an uncertainty factor of 1UO, was calculated to be 0.67 mg/L. Since this value
is much higher than the One-day HA value (0.18 mg/L) for a 10-kg child, it was
not considered adequate.
3. Longer-term Health Advisory
)
The study by Bainova (1969) has been selected to serve as the basis for
the Longer-term HA values for children and adults. Rats were administered
diquat orally for 4-1/2 months at 2.1 and 4.3 mg/kg bw/day (1.1 and 2.3 mg
diquat ion/kg bw/day). Biochemical and histological changes were seen at botn
doses; the higher dose produced more severe effects. Although there was an
apparent dose-response relationship, statistical significance was reported at
the high dose only; effects at the low dose were not documented. Diquat produced
elevated hematopoieses and changes in indices associated with liver function.
Histological changes in the lungs, liver, and testes were found. The most
characteristic feature was lung damage characterized by papillomatous proliferations
of the bronchial and bronchiolar epthelia. Statistically significant clinical
effects were reported for the high dose, however, the data for the low dose was
not documented. There was no clear data base to consider the low dose as a LGAEl
or NOAEL, therefore, 1.1 mg/kg bw/day was determined to be the Low Effect Level
(LEL) in rats.
Using tnis study, the Longer-te"m HA for a 10-kg child is calculated as
follows:
(1.1 ma/kg bw/day)(10 kg) = O.j4 ^ ai^uat ion/L (40 ug/L)
U L/day)(3UU)
VII1-9
-------
where:
1.1 mg/kg/day
LEL, based on histological and biochemical chanyes in rats]
administered diquat orally for 4,5 months (Bainova et
al., 1969).
I
10 kg = assumed weight of a child.
1 L/day = assumed water consumption of a 10-kg child.
3UU = uncertainty factor, chosen in accordance with NAS/OOw
guidelines for use with a LOAEL from an animal study.
A three-fold uncertainty factor was used instead of trie ;'
standard 10 used with a LOAEL since the dose was conside~eb
to be a LEL in the absence of an adequate database to clas'sif.
it as a NOAEL or LOAEL. This uncertainty factor was used!'
to compensate for the quality of information from this study
since the LEL may or may not have been adverse.
The Longer-term HA for a 70-kg adult consuming 2 L of water per day is
calculated as follows: ,
(1.1 mg/kg/day)(70 kg)
(2 L/day)(300)
= U.13 mcj diquat ion/L (rounded to 100 ug/L)
where:
1.1 mg/kg/day
70 kg
2 L/day
3UO
LEL, based on histological and biochemical changes in rats;
administered diquat orally for 4-1/2 months (Bainova et
al., 1969).
assumed weight of an adult.
assumed water consumption of an adult.
uncertainty factor, chosen in accordance with NAS/OUv4*""**^
guidelines for use with a LOAEL from an animal study.
A three-fold uncertainty factor was used instead of cne
standard 1U used with a LOAEL since the dose was considered
to be a LEL in the absence of an adequate database to class if
it as a NOAEL or LOAEL. This uncertainty factor was used '
to compensate for the quality of information from trrs sdjdy
since the LEL :nay or may not have been adverse.
No existing guidelines or stanaa-as *e~e found for longer term (subch-onici]
oral or inhalation exposure to diquat.
VIIl-10
-------
4. Reference Dose and Drinking Water Equivalent Level
Table VIII-2 summarizes the studies considered for derivation of the RfO
and OWEL for diquat. The study by Pi Me and Rees (1970) on rats was not selec-
ted because the lowest dose used (13 mg diquat ion/kg/day) resulted in cataract
development. A study by Bainova and Vulcheva (1978) gave a LOAEL of 2 mg
diquat ion/mg/day in rats. This study was not selected because other studies
with NOAELs were available- for calculation of the DWEL. The 1-year studies by
Makouskii (1972) on rats and guinea pigs were taken from a secondary source and
detailed documentation was not available. As such, these studies were not
selected. The only chronic study with dogs (Clark and Hurst, 1970) was not
selected because a number of rat studies with much lower NOAEL values were
available. NOAEL values ranging from 0.2 to 1.25 were identified from three
dietary chronic studies with rats (Rogerson and Broad, 1978; Colley et al.,
1985; Clark and Hurst, 1970). The study by Rogerson and Broad (1978) was not
selected because the NOAEL of 1.25 mg diquat ion/kg/day exceeded the LOAEL of
1.1 mg diquat ion/kg/day identified from the Clark and Hurst (1970) study.
The 2-year feeding study in rats conducted by Colley et al. (1985) has
been selected to serve as the basis for the RfD and DWEL. The NOAEL in this
study (based on absence of cataract formation) was 0.22 mg/kg/day. This value
is much lower than the results of Rogerson and Broad (1978), who reported a
NOAEL (also based on absence of cataract formation) of 1.25 mg/kg/day in a
2-year feeding study in rats. The NOAEL is supported by the data from the study
by Clark and Hurst (1970) which also established 0.22 mg/kg/day as the NOAEL
in rats from a 2-year feeding study.
Assumptions regarding absorption a"* not required in the DWEL calculation
because the NOAEL is expressed in :e~is of an intake dose rather than an aosorDed do:
VIII-11
-------
Table VIII-2. Summary of Candidate Studies for
Derivation of the DUEL for Diquat
~im
Reference
Clark and
Hurst (1970)
Clark and
Hurst (1970)
Rogerson and
Broad (1978)
Pirie and
Rees (1970)
Makovski 1
(1972)
Makovski i
(1972)
Bainova and
Vulcheva
(1978)
Col ley et al .
(1985)
Species
Rat
Doy
Rat
Rat
Rat
Guinea
pig
Rat
Rat
Route
Diet
Diet
Diet
Diet
Oral
Oral
Drinking
water
Diet
Exposure
Duration
2yr
2-4 yr
2 yr
70 wk
1 yr
1 yr
1-2 yr
2 yr
Endpoints
Cataract
development
Cataract
development
Cataract
development
Cataract
development
..a
--
Lung
histology
Cataract
development
NOAEl
(mg diquat
ion/kg/day)
0.22
1.2
1.25
0.2
0.1
«* <
0.22
LOAEL j
(mg diquat
ion/kg/lay)
li
1.1.;
3.6;!
3.7iJ
!|
13
-- '
"" ii
0.65J
h
,|
reported in the secondary source,
VI 11-12
-------
Using this study, the DWEL is derived as follows:
Step 1: Determination of the Reference Dose (RfD)
RfO = (U.22 mg/kg/day) s U.OU22 mg diquat ion/kg/day
where:
0.22 mg/kg/day = NOAEL following 2-year oral exposure in rats (Col ley et
al., 1985).
1UO = 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 (DWEL)
DWEL - (U.U022 mg diquat 1on/kg/day)(7U kg) » 0.077 mg diquat ion/L (rounded
. ., y H V
where:
0.0022 mg diquat ion/kg/day - RfO.
70 kg » assumed weight of a 70-Kg adult.
2 L/day « assumed water consumption of an adult.
This OWEL calculation assumes that 100% of the human exposure is derived
from drinking water. This value may be modified upon the availability of
relative source contribution data that provide human exposure estimates from
food, air, and possibly the occupational environment. >-*
C. QUANTIFICATION OF CARCINOGENIC .EFFECTS FOR DIQUAT
Table VIII-3 summarizes the studies considered for derivation of carcino-
genic risk estimates. In four 2-year feeding studies with rats and one 1-year
feeding study with mice, animals were administered daily doses of up to 75 mg
diquat ion/ky bw, no evidence of carcinogenic activity was reported (Rogerson
VIII-13
-------
and Broad, 1978; Clark and Hurst, 1970; Bainova and Vulch'eva, 1978; Colley et
al., 1985; Ben-Dyke et al., 1975). However, the conclusions from the studies
by Clark and Hurst (1970) and Ben-Dyke et al . (1975) require additional
information before a complete evaluation of these studies can be made. No ,j
risk assessment has been performed at the present time because no evidence of ;
i
carcinogenic activity has been found. The International Agency for Research on-
Cancer (WHO, 1982) and EPA have not evaluated the carcinogenic potential of \
diquat. '
1
D. SUMMARY
i
The recommended values for the One-day HA for a child, the Longer-term HAsij
for both children and adults, and the DUEL are summarized in Table VIII-4.
VI11 -.14
-------
'Table VIII-3. Summary of Candidate Studies for Derivation of
the Carcinogenic Risk Estimates for Diquat
Reference
Exposure
Species Route duration
Results
Rogerson and Broad
(1978)
Clark and Hurst
(1970)
Bainova and
Vulcheva (1978)
Col ley et al. (1985)
Ben-Dyke et al.
(1975)
Rat
Rat
Rat
Rat
Diet
Diet
Oral
Diet
Mouse ' Diet
2 yr No evidence of induced tumors
2 yr No evidence of induced tumors
1-2 yr No evidence of malignancy .
104 wk No carcinogenic effects
(incomplete data)
80 wk No treatment-related incidence
of neoplasms
(incomplete data)
VIII-15
-------
Table VIII-4. Summary of Quantification of Toxicoloyical Effects for Diquat
. Value
One-day HA (10-ky child)
Ten-day HA (10-kg child)
Longer-term HA (10-kg child)
Lonyer-term HA (7U-kg adult)
DUEL (70-kg adult)
Excess cancer risk (10-6)
Drinking water
concentration
(ug diquat ion/L) Reference
300 ' Crabtree et al . (1977)
V
..a
40 Bainova et al . (1969)
10U Bainova et al. (1969)
80 Col ley et al . (1985)
._
Longer-term HA value for a 10-kg child is taken as a conservative esti-
mate of the Ten-day HA value.
VIII-16
-------
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