• 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

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                              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

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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

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     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

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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

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     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

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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

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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

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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

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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

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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

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                  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

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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

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     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

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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

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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

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 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

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         .  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

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        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

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               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

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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

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        '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

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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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