United States'
                    Environmental Protection
 Health Effects Research
 Research Triangle Park NC 27711
                    Research and Development
 EPA/600/S1 -86/003  June 1986
&EPA         Project Summary

                    Carcinogenic  Potential of
                    Arsenic  Compounds  in
                    Drinking Water

                    Donald Y. Shirachi, Shing-Hui Tu, and John P. McGowan
                     The primary objective of this research
                    study was to determine whether arsen-
                    ite (Aslll), arsenate (AsV), dimethyl-
                    arsinic (DMA) and monomethylarsonic
                    acid (MA) were initiator carcinogens or
                    promoters of DENA-initiated tumors in
                    the rat liver.
                     The maximum tolerated doses (MTD)
                    of the arsenics to be used in this study
                    were determined by treating male Wis-
                    tar rats with increasing drinking water
                    concentrations ranging from 10 ppm -
                    2560 ppm for 7 weeks, depending upon
                    the arsenic studied. The apparent MTD
                    determined were: MA, 800 ppm; Aslll,
                    160 ppm; AsV. 160 ppm; and DMA, 80
                    ppm. The calculated LD50 were DMA,
                    98.5  ppm;  ASIII,  264.8 ppm; AsV,
                    294.5 ppm; and MA, 1160.1 ppm.
                     To determine if those arsenic com-
                    pounds acted as an initiator or promoter,
                    male Wistar rats were partially hepatec-
                    tomized, treated with a single dose of
                    diethylnitrosamine (30 mg/kg i.p.); on
                    day 7 treatment began with the MTD of
                    each arsenic for 7, 25, and 43 weeks in
                    the drinking water. This was the pro-
                    moter protocol. The initiator protocol
                    did not include the diethylnitrosamine
                    treatment. There were no significant
                    numbers of animals with tumors in the
                    liver in any of the arsenic treated animals
                   as compared to their controls  at the
                   three time periods of treatment with
                   either the initiation or promotion proto-
                     However, in the promotion protocol
                   there was significant increase (p < 0.05)
                   in the number of animals exhibiting
                   tumors in the kidney in the Aslll pro-
                   moted group, 7/10, as compared to the
                   DENA controls, 2/9 when treated for
 25 weeks. In both protocols Aslll treat-
 ed animals showed a significant de-
 crease in thymus gland weights. This
 was suggested to be a contributory
 factor in the renal tumorigenesis ob-
 served in this study.
   This Project Summary was developed
 by EPA's Health Effects Research Lab-
 oratory, Research Triangle Park, NC. to
 announce key findings of the research
 project that is fully documented in a
 separate report of  the same title (see
 Project Report ordering information at

  The final report presents the results of
 three years of study to determine whether
 arsenite (Aslll), arsenate (AsV), dimethyl-
 arsinic acid (DMA),  and monomethylar
 sonic acid (MA) were  initiator carcinogens
 or promoters of tumors in the rat liver.
 This work was initiated to gain data that
 might clarify some of the apparent contra-
 dictions between human and animal data
 and provide the basis for arriving at more
 realistic estimates of the carcinogenic
 risk that is associated with the various
 forms of arsenic found in drinking water.
 A brief review of literature illustrates the
 extent of the contradictory data.
  Within the past several years many
 researchers have investigated the rela-
tionship between arsenic exposure and
development of cancerous lesions in
 humans; others have sought to corrob-
orate those findings in animal experi-
ments as explained in the  next two
 The relationship between arsenic ex-
posure and the development of cancerous
lesions in humans has been reviewed by

IARC (1973), Yeh (1973), Zaldivar (1974),
Bencko (1976),  Committee (1977),  and
Wildenberg (1978). They cite many clin-
ical and  epidemiological  observations
which  indicate that when humans are
exposed to arsenic, via dietary, medical,
environmental or  occupational routes,
neoplasms of the liver, lung, skin, lym-
phatic and hematopoietic tissues result.
Hueper (1953)  referred to arsenic as
"after coal soot, the oldest known human
carcinogen." Dinman  (1974) placed ar-
senic,  along  with  mustard gas,  in  a
"definitely carcinogenic" category based
on epidemiological evidence for "carcino-
genic potential."
  However, not one of the 46 arsenic
exposure experiments listed in the "Sur-
vey of  Compounds Which Have Been
Tested  for Carcinogenic Activity," con-
clusively demonstrated arsenic's carcino-
genic potential (Hartwell, 1951; Shubik
and Hartwell, 1957). Nor have any recent
studies demonstrated arsenic carcino-
genesis in animals (Wildenberg, 1978).
So, even though epidemiological evidence
shows  arsenic  exposure  is related to
human cancers, attempts to induce can-
cer experimentally with arsenic com-
pounds have not been successful. Even in
the short-term mutagenicity tests, arsenic
compounds (like other  metals) have given
negative results (Wildenberg, 1978).
  Two  of the reasons why data from
experimental animals  have not demon-
strated a positive  correlation  between
arsenic exposure and carcinogenesis are:

  1.  The route of administration was via
     the diet or by application to the skin.
     Arsenic, unlike other skin carcin-
     ogens, does not  exert its action by
     direct contact. There are  no docu-
     mented cases of human skin can-
     cers following the external applica-
     tion of medicinal arsenicals, nor is
     there evidence that arsenic in food
     acts as a human carcinogen. Man's
     primary  routes of arsenical expo-
     sure resulting in cancer  are from
     ingestion in aqueous and alcoholic
     (in wines) solutions and inhalation
     (Committee, 1977). Schrauzer et al.
     (1978) have reviewed reports which
     indicate inorganic arsenic com-
     pounds have different toxicities and
     interactions with selenium when
     animals drink rather than eat their

  2.  In general, a single concentration of
     only one  arsenic compound  was
     tested and the maximum tolerated
     dose (MTD) was not determined.
     leading to poor animal survival. Too
     few tumors were produced per
     experimental  treatment group  to
     allow a definitive statement  con-
     cerning arsenic carcinogenesis.
     Even if the number of animals per
     test were increased substantially,
     only those chemicals which cause
     high tumor incidence would readily
     be  detected as carcinogens. But
     observations  in humans  indicate
     arsenicals are probably weak car-
     cinogens (Wildenberg, 1978).

       Therefore, what is needed are bio-
     assays which have greater sensi-
     tivity to arsenic than do either the
     classical long-term feeding studies
     or the short-term microbiological
     mutagenicity tests. We propose to
     explore the use of an assay which
     uses a two-stage carcinogen treat-
     ment to demonstrate experimental-
     ly the carcinogenic potential of four
     different arsenic compounds.
Materials and Methods

The Maximum Tolerated
Dose (MTD) and the LD50 of
Arsenic Compounds in
Wistar Pats Exposed to
Arsenic Compounds in the
Drinking Water for 7 Weeks
  Male Wistar rats (60-80 g), obtained
from Simonsen Laboratories, Inc. (Gilroy,
CA) were randomly divided into groups of
five rats each and given access to distilled
water containing various parts per million
(ppm) arsenic as either sodium arsenite
(Aslll); sodium arsenate (AsV); methyl-
arsonic acid, disodium  salt (MA); or
dimethylarsinic acid, sodium salt (DMA)
for seven weeks. Each group was housed
in individual gang cages and fed a stand-
ard laboratory chow. The weights of all
test animals were recorded weekly. Gross
observations were made during the test
period. At 7 weeks, the animals  were
sacrificed by guillotine  and routine
necropsy was performed.

The Stimulation of Gamma-
Activity in Rat Liver by
Inorganic Arsenics
  Five-week old Male Wistar rats ob-
tained from Simonsen Laboratories, Inc.
(Gilroy, CA) were  randomly divided into
groups  of five rats  each, housed in
individual gang cages and given access to
standard laboratory  rat chow and  to
distilled water or distilled water contain-
ing 160 ppm (elemental arsenic) of either
sodium arsenite (Aslll) or sodium arsenate
(AsV). The  animals were sacrificed  at
predetermined times  by guillotine. The
liver tissue was quickly excised and
prepared for histochemical and biochem-
ical studies.
The Initiator Potential of
Arsenite and Arsenate in
Partially Hepatectomized
Rats When Promoted by
  Male Wistar rats weighing 70-100 g
were 2/3 partially hepatectomized (PH)
and 18-24 hours later were exposed to
arsenic in their drinking water for a period
of 3 days. The Aslll and AsV doses were
160 ppm (MTD) and 320 ppm. The higher
dose was given in order that the concen-
tration of As in the plasma and liver would
be comparable to doses  in animals ex-
posed to  160 ppm  As for one week.
Previous experiments indicated that 1
week was the minimum period of time
necessary for GGTase stimulation at 160
ppm. Animals were then given 0.05%
phenobarbital food pellets (Teklad Test
Diets) ad libitum 7 days after the pH and
continued for 7 weeks. The animals were
sacrificed by guillotine; livers were dis-
sected out and processed as previously
described in Section 4 of the full report;
and routine necropsy was performed.
Promoter Potential of
Arsenic Compounds in
Partially Hepatectomized
DEN A -Initiated Rats
  Male Wistar rats weighing 70-100 g
were partially hepatectomized (PH) and
injected i.p. 18-24 hrs later with DENA
(Eastman Kodak) at a dose of 30 mg/kg.
On day 7 the animals were treated to one
of the following:  0.05% phenobarbital
(PB) in the diet, arsenite (Aslll) or arsenate
(AsV) present in the drinking water at a
level of 160 ppm, or organic arsenicals,
DMA  and  MA, present in  the drinking
water at a concentration of 80 ppm and
800 ppm, respectively. Each group con-
tained a minimum of five animals. At the
end of 7 weeks of treatment with PB or
the arsenics the animals were sacrificed
by guillotine and routine necropsy per-
formed. Liver samples were removed for
GGTase assay.

Promoter Potential of
Arsenic in Partially
Hepatectomized DENA-
Initiated Rats
  In this study, sham-operated animals
were included as  control treatment
groups for the promotion experiments.

Promoter Potential of
Arsenic in Partially
Hepatectomized DENA-
Initiated Rats
  For animals in this study, the water
intake for each cage was monitored daily.
From this data daily doses for the arsenics
were calculated. At the scheduled sacri-
fice days, both kidneys  were removed
from each animal, weighed and processed
for histochemical, biochemical and histo-
logical examinations.

Conclusions and
  The experimental design for this study
which was the broad spectrum initiation-
promotion protocol developed at HERL-
Cincinnati.lt was effective in determining
whether a specific arsenic compound had
an  initiator carcinogen  potential or a
promoter potential. Although the proce-
dure was designed for testing potential
initiators and/or promoters in the liver,
the authors believe it can be used to study
potential agents in the kidney.
  The study also demonstrated that the
chronic toxicity of arsenic compounds in
drinking water cannot be predicted from
acute toxicity studies. An organic arsenic
such as dimethylarsinic acid was as toxic
as arsenite or arsenate when adminis-
tered chronically in  drinking water. Ac-
cording to acute toxicity studies arsenite
and arsenate are methylated to dimethyl-
arsinic acid which is supposed to be an
inactive metabolite. There appears to be a
discrepancy between the results of the
acute toxicity study and the results. The
results of this study showed that metab-
olism would result in an equitoxic metab-
olite. Thus,  it is  clear that the toxicity of
these compounds have to be reevaluated
under chronic exposure conditions  in
order to determine the effect of metabo-
lism on steady-state levels of the metabo-
  The study demonstrated that arsenite
is a promoter in  the kidney. Chronic
arsenite treatment for 25 weeks promoted
DENA-initiated tumors in the kidney. As a
result, arsenite  may now be used as  a
known promoter in the kidney to test for
potential initiator carcinogens.
  We have also demonstrated that arse-
nite decreases the thymus weight which
is suggestive of a decrease in the immune
response. Thus, this immunosuppressive
effect may be a contributory factor in the
carcinogenesis process.
  Because these experiments were per-
formed with partial hepatectomy, it must
yet be determined  that this surgical
procedure did not contribute to the pro-
motion effect of arsenite. Experiments
should be performed on intact animals
under the same conditions of dosing. It is
anticipated that the treatment time and
the time for appearance of renal tumors
will be extended.
  Since the arsenite concentration used
in this study was the maximum tolerated
dose, a dose-respose study should  be
done to determine at what level arsenite
no longer has promoter activity.  This
would be important for setting safe
drinking water standards.
  Because of the discrepancies found
between the results obtained from chron-
ic dosing in the drinking water as com-
pared to the results found in the literature
from acute toxicity studies, the authors
recommend that  metabolism of the dif-
ferent arsenics be studied in vivo chronic
dosing in the drinking water. This would
also be important in setting safe drinking
water standards.
  Finally, the immunosuppressive effect
of arsenite should be studied further to
shed some light on the role of arsenite in
the chemical carcinogenesis process.

Bencko, V.,  B. Benes,  and M. Cikrt,
Biotransformation of As(lll) to As(V) and
ArsenicTolerance. Arch. Toxicol., 36:159

Committee on Medical and Biologic Ef-
fects of Environmental Pollutants. Arse-
nic. National Academy of Sciences. Wash-
ington, D.C. pp. 332 (1977).

Dinman, D. B., The Nature of Occupational
Cancer. CC. Thomas, Springfield, IL, pp.
101 (1975).

Hueper, W. C. Environmental Carcino-
genesis in Man and Animals. Ann. N.Y.
Acad. Sci., 108:963(1963).

Hartwell, J.  L, Survey of Compounds
Which Have Been Tested for Carcinogenic
Activity, 2nd ed. DHEW; Publication No.
(NIH) 72-35.  PHS Publication  No.  149.
U.S. Government Printing Office, Wash-
ington, D.C. pp. 583 (1951).
IARC Monographs on the Evaluation of
the Carcinogenic Risk of Chemicals to
Man. Vol. 2. Some Inorganic and Organo-
metallicCompounds. Lyonpp. 181 (1973).

Wildenberg, J., An Assessment of Experi-
mental Carcinogen-Detecting System
with Special Reference to Inorganic Ar-
senicals. Environ. Res.  16:139(1978).

Yeh, S., Skin Cancer in Chronic Arseni-
calism. Human Pathol. 4:469 (1973).

Zaldivar,  R.  Arsenic Contamination of
Drinking Water and Food Stuffs Causing
Endemic Chronic Poisoning. Beitr. Path.
Bd., 151:384(1974).

    Donald Y. Shirachi, Shing-Hui Tu, and John P. McGowan are with the University
      of the Pacific, Stockton. CA 95211.
    Merrel Robinson is the EPA Project Officer (see below).
    The complete report, entitled "Carcinogenic Potential of Arsenic Compounds in
      Drinking Water." (Order No. PB 86-200 854/AS; Cost: $11.95, subject to
      change) will be available only from:
            National Technical Information Service
            5285 Port Royal Road
            Springfield, V'A 22161
            Telephone: 703-487-4650
    The EPA Project Officer can be contacted at:
            Health Effects Research Laboratory
            U.S. Environmental Protection Agency
            Research Triangle Park, NC 27711
United States
Environmental Protection
Center for Environmental Research
Cincinnati OH 45268
Official Business
Penalty for Private Use $300

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