NCEA-W-0372
March 1998
DICHLOROACETIC ACID
Carcinogenicity Identification Characterization Summary
National Center for Environmental Assessment-Washington Office
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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CONTENTS
LISTS OF TABLES AND FIGURES . . iv
PREFACE : v
AUTHORS, CONTRIBUTORS, AND REVIEWERS vi
1. INTRODUCTION : : 1
2. PREVIOUS EVALUATIONS OF CARCINOGENICITY 2
2.1. PRIOR EPA ANALYSES 2
2.2. RECENT IARC MONOGRAPH 2
2.3. 1997 ILSI REPORT ._ 3
3. METABOLISM OF DCA ". . . 3
4. STUDIES OF DCA CARCINOGENICITY , 5
4.1. HUMAN DATA 5
4.2, DCA CARCINOGENICITY BIOASSAY DATA IN RATS AND MICE 7
4.2.1. Liver Tumors in Animals—EPA Guidelines Position 17
4.2.2. Sex Differences '. 18
5. MODES OF CARCINOGENIC ACTION THAT MAY RELATE TO LIVER TUMORS . 18
5.1. MUTAGENICITY AND GENOTOXIC EFFECTS 19
5.1:1. Evidence for DCA Mutagenic Potential 19
5.1.2. Information Regarding Mutation Spectra in DCA-Induced Tumors 20
5.1.3. Summary 21
5.2. PEROXISOME PROLIFERATION 22
5.3. EFFECTS'ON THE INSULIN RECEPTOR 23
5.4. ALTERATION IN DNA METHYLATION 23
5.5. ALTERATIONS IN CELL REPLICATION AND DEATH RATES 24
5.6. CYTOTOXICITY AND COMPENSATORY HYPERPLASIA 25
5.7. HEPATOMEGALY 26
6. THE ISSUE OF DOSE LEVEL AND MTD '...- 27
7. SUMMARY AND CONCLUSIONS 29
8. REFERENCES 31
APPENDIX A: Charge to Peer Reviewers and Comments A-l
APPENDIX B: Remarks on Peer-Review Comments , •. B-l
in
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LIST OF TABLES
1. Animal cancer bioassays for dichloroacetic acid 8
LIST OF FIGURES
1. Proposed metabolic scheme for dichloroacetate 6
IV
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PREFACE
This document, prepared by the National Center for Environmental Assessment-
Washington Office, responds to a request from the EPA's Office of Science and Technology,
Office of Water, for a brief hazard characterization regarding the potential carcinogenicity of
I *
dichloroacetic acid (DCA) in humans.
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
This document was prepared by the National Center for Environmental Assessment-
Washington Office (NCEA-W).
AUTHOR
Jean C. Parker, Ph.D., NCEA-W/ORD
CONTRIBUTOR
Robert McGaughy, Ph.D., NCEA-W/ORD
EPA REVIEWERS
Krishan L. Khanna, Ph.D., OW
Penelope Fenner-Crisp, Ph.D., OPPTS
Robert McGaughy, Ph.D., ORD
Martha Moore, Ph.D., ORD
Steve Nesnow, Ph.D., ORD
Cheryl Siegel-Scott, M.S., ORD
Vicki Vaughn-Dellarco, Ph.D., OW
Vanessa Vu, Ph.D., OPPTS
Jeanette Wiltse, Ph.D., OW
EXTERNAL PEER REVIEWERS
R. Julian Preston, Ph.D.
Chemical Industry Institute of Toxicology
Research Triangle Park, NC
James A. Swenberg, D.V.M., Ph.D.
University of North Carolina
Chapel Hill, NC
Lauren Zeise, Ph.D.
California EPA
Berkeley, CA
ACKNOWLEDGMENT
Special .thanks to Terri Konoza of NCEA-W's Operations and Support Group, Technical
Information Staff, for her dedication and document production assistance.
vi
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1. INTRODUCTION
This document, prepared by the National Center for Environmental Assessment-
Washington Office, responds to a request from EPA's Office of Water for a brief hazard ,
characterization regarding the potential carcinogenicity of dichloroacetic acid (DCA) in humans.
The objectives of this report are to develop a weight-of-evidence characterization, in the spirit of
EPA's 1996 Proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1996), for the
potential human carcinogenic hazard posed by DC A, as well as to provide a response to certain
issues raised by an expert panel of the International Life Sciences Institute (ILSI) in their report
An Evaluation of EPA's Proposed Guidelines for Carcinogen Risk Assessment Using
Chloroform and Dichloroacetate as Case Studies: Report of an Expert Panel (ILSI, 1997).
This hazard characterization relies on information available in existing peer-reviewed
source documents and certain key scientific publications. The current assessment addresses
meaningful issues important to interpretation of DC A carcinogenicity data, particularly regarding
mechanistic information pertinent to the etiology of DC A-induced rodent liver tumors and their
relevance to humans. The paper also speaks to study design issues and concerns that must be
dealt with in interpreting and understanding the human relevance of the induced
hepatocarcinogenicity observed in rats and mice. In developing this narrative summary,
emphasis is thus placed on information that has bearing on the relevance of the animal
i
carcinogenicity data and key lines of evidence that contribute to the overall weight of evidence
concerning the potential human carcinogenicity of DCA. Furthermore, this paper discusses the
level of uncertainty associated with the relevance of the data to the human situation. It is
important to realize that this characterization addresses the overall weight of evidence for a DCA
human cancer hazard from a qualitative perspective only. The available published studies are not
considered adequate to support biologically based quantitative dose-response estimation at low
doses. Quantitative estimates of risk are not necessary for establishing a maximum contaminant
level goal (MCLG) for drinking water. It is also important to recognize that this paper is a
hazard characterization conclusion and is not a traditional, self-contained, indepth health
assessment document. The bulk of the specific data is discussed in other documents and reports.
Three scientists provided an outside expert peer review of the February 12; 1998,
External Review Draft of this paper. Several EPA scientists also critiqued the paper at various
stages during its development. Each of the comments and suggestions was carefully considered,
resulting in changes to the paper where appropriate. The final draft is an improved product
because it addresses important points raised by reviewers and incorporates many of their
1
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thoughtful suggestions. Although the comments of the external peer reviewers are generally
favorable in acknowledging that the paper is well written and that it adequately discusses the
important relevant data on mode of carcinogenic action and many of the issues surrounding
potential DCA cancer hazard, these reviewers do not necessarily agree with EPA on all the
controversial points. In fact, the expert reviewers do not agree with each other on certain key
items due to the polemic nature of certain of the issues. One of the three peer reviewers agrees
with the EPA's bottom line conclusion regarding potential human hazard—that DCA is likely to
be a human carcinogen. Another reviewer believes that it is premature at this, time to come to the
EPA's conclusion, while the third reviewer does not specifically agree or disagree with the
EPA's hazard conclusion. The written reviews officially submitted to EPA by the three external
scientists are presented as Appendix A.
2. PREVIOUS EVALUATIONS OF CARCINOGENICITY
2.1. PRIOR EPA ANALYSES
The Office of Water published a criteria document in 1994 for haloacetic acids, including
DCA, formed as by-products of chlorine disinfection of drinking water. An Integrated Risk
Information System (IRIS) carcinogenicity summary was developed for DCA and updated in
1996 (U.S. EPA, 1998). These reviews placed DCA into Group B2 (probable human
carcinogen) in accordance with, the 1986 EPA Guidelines for Carcinogen Risk Assessment (U.S.
EPA, 1986). The DCA categorization was based primarily on findings of liver tumors in rats and
mice, which was regarded as "sufficient" evidence in animals. This classification referred only
to the weight of the experimental evidence that DCA may cause cancer in humans and not to the
potency of its carcinogenic action.
2.2. RECENT IARC MONOGRAPH
In 1995, the International Agency for Research on Cancer (IARC) reviewed and evaluated
the available published data relevant to DCA carcinogenicity. It is noteworthy that the IARC
•
working group evaluated a different database from the one assessed by EPA (and therefore came
to a different conclusion). Because EPA bioassay studies in F344 rats were not fully published
until after the IARC working group meeting (DeAngelo et al., 1996; Richmond et al., 1995),
these studies were not included in the 1995 IARC monograph. Positive findings in female mice
(Pereira, 1996; Pereira and Phelps, 1996) were not available at that time, either. Thus,
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observation of increased liver tumors in male B6C3F, mice (Herren-Freund et al., 1987; Bull et
al., 1990; DeAngelo et al., 1991; Daniel et al., 1992) was the only DCA tumor evidence
reviewed, and the evidence in animals was considered to be "limited." Therefore, the IARC
working group concluded that "DCA is not classifiable as to its carcinogenicity to humans,"
placing DCA in the IARC Group 3 category.
2.3. 1997 ILSI REPORT
Recently, ILSI convened a panel of experts for the purpose of evaluating EPA's 1996
Proposed Guidelines for Carcinogen Risk Assessment using chloroform and DCA as case studies
(ILSI, 1997). The final report (November 1997) of this panel identifies issues associated with the
application.of the proposed guidelines. It also presents a qualitative assessment of cancer hazard
posed by DCA, concluding that the carcinogenicity of DCA cannot be determined, on the basis of
a lack of carcinogenicity evidence in humans and inadequate data in experimental animals.
Views expressed in the report are stated to be those of individual expert panel members and do
not necessarily represent those of their respective organizations or ILSI.
3. METABOLISM OF DCA
DCA is biotransformed in the body, and its metabolism is a saturable process. It is not
known whether the parent DCA compound, a metabolite, or DCA in concert with certain of its
metabolites is the putative chemical species in tumorigenesis. It is thought, however, that the
DCA compound itself, unlike some other chlorinated molecules, contributes directly to the
process of tumor induction. There are, however, postulated reactive intermediates that may bind
to macromolecules and contribute to tumor induction. Qualitatively, DCA metabolism is similar
in rats, mice, and humans. Repeated dosing of DCA results in inhibition of its own metabolism
in humans at therapeutic doses as well as in rodents at bioassay doses (Curry et al., 1985,1991;
Henderson et al., 1997; Comett et al., 1997; Gonzalez-Leon and Bull, 1996; Gonzalez-Leon et
al., 1997a, 1997b), a fact that may be important at lower doses in allowing a relatively higher
percent dose of parent compound to interact with the target tissue relative to higher doses. On
the other hand, if the inhibition of DCA metabolism is due to saturation of a metabolic
conversion of a DCA metabolite, or to a depletion of an enzyme or required cofactor, the
phenomenon may be limited to DCA doses higher than those usually found in chlorinated
drinking water. If this is the case, a greater proportion of parent compound would be
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biotransformed at the low doses, giving a relatively higher percentage of intermediate metabolite
to interact with target tissue. Theoretically, a greater proportion of parent compound could
initially be metabolized to an intermediate, followed by a relatively greater percentage of parent
compound metabolism being inhibited. Whether lower doses are relatively more, or less,
potency would depend on whether the parent compound, or its intermediate metabolites, or both,
can be involved in tumor induction and their respective modes of action.
The biotransformation of DC A has been evaluated in animals in vivo and in vitro as well
as in clinical studies in humans (Curry et al., 1985, 1991; Stacpoole, 1989; Stacpoole et al., 1990,
1992; Larson and BuH, 1992a, b; Stevens et al., 1992; Lin et al., 1993; Templin et al., 1993,
1995; Lipscomb et al., .1995; Gonzalez-Leon and Bull, 1996; Gonzalez-Leon et al., 1997a, b;
James et al., 1997; Stacpoole, 1998; Tong et al., 1998). DCA is well absorbed orally and is
rapidly cleared from the systemic circulation. This effective clearance from blood and a
relatively short half-time may indicate high target organ concentrations, with binding to tissue, or
accelerated metabolism, or high affinity for both processes. The plasma half-time increases
significantly with repeated dosing in animals (Gonzalez-Leon et al., 1997b, 1998), as well as
multiple doses in humans (Curry et al., 1985, 1991). This implies that information from single-
dose .studies is probably not useful for interpreting internal dose levels in chronic bioassays.
DCA metabolic products are the same in rodents and humans.
Stacpoole et al. (1990) proposed a pathway involving reductive dechlorination of DCA to
form monochloroacetate (MCA), and eventually thiodiacetic acid via glutathione conjugation. In
addition to the biotransformation pathway for chlorinated acetates proposed by Stacpoole et al., a
second metabolic pathway for DCA was proposed by Larson and Bull (1992a)—enzymatic
hydroxylation of the C-H bond, with spontaneous dehydrodechlorination to a reactive acid
chloride intermediate that is expected to rapidly hydrolyze to oxalic acid. Although Larson and
Bull believed this reaction to be microsomal P450-mediated, Lipscomb et al. (1995) have
demonstrated more recently that dehalogenation occurs primarily in cytosol in the presence of
NADPH^and GSH. Glyoxylate has been shown to be the chief biotransformation product from
this pathway (Comett et al., 1997; James et al., 1997). Anders and his coworkers (Tong et al.,
1998) have since identified and characterized a rat liver cytosolic glutathione transferase Zeta
that catalyzes the conversion of DCA to glyoxylate. The characteristics of this enzyme closely
match those of the newly discovered human glutathione transferase Zeta (Board et al., 1997).
These investigators (Tong et al., 1998) postulate the formation of a reactive intermediate
metabolite that may bind to protein. Formation of a metabolite such as a glutathione-bound
MCA with a reactive chlorine, or a hemithioacetal intermediate, which binds covalently to
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macromolecules. may explain the ability of DC A to inhibit its own metabolism through apparent
enzyme inactivation, as has been shown in vivo in rats and mice (Gonzalez-Leon et al., 1997b ),
in vitro in rat liver cytosol (Gonzalez-Leon et al., 1998), and in humans (Gurry et al., 1985). If
this reactive intermediate can reach the nucleus, then a novel kind of DNA adduct could be
formed (glutathione-acetic acid nucleotide). This is similar to what has been proposed for
dichloromethane (Hashmi et al., 1994). Free radical formation in the reductive dechlorination
pathway has been previously implicated as a possibility for DNA damage through strand breaks
and cross links, potentially leading to gene mutation or chromosomal aberrations (Chang et al.,
1992). Now there is new information indicating that the oxygenation-of-DCA-to-glyoxylate
process may also produce at least one reactive intermediate that could bind to protein and
possibly form DNA adducts. Therefore, if DC A rodent hepatocarcinogenicity is related to
glutathione transferase Zeta-dependent bioactivation of the parent compound, and analogous
biotransformation occurs in humans, this has important implications for human hazard. Similar
rates of DCA biotransformation are reported by Tong et al. (1998) for purified rat liver and
recombinant human enzymes. Elimination half-times have been shown to be similar in rats and
humans (James et al., 1997).
The glyoxylate metabolite is further processed via at least four routes. It is converted to
oxalate, glycolate, carbon dioxide, and glycine, all of which have been identified as DCA
metabolites (ILSI, 1997). Recently, Comett et al. (1998) identified one of three unknown urinary
metabolites of DCA in rat as hippurate. These investigators concluded that glyoxylate formed by
cytosolic DCA dechlorination is transaminated to glycine, which is then conjugated with benzoic
acid to give hippurate, a final excreted metabolite. A schematic DCA biotransformation is
depicted in Figure 1. This proposed metabolic pathway for DCA is based on the findings
reported by the investigators cited in this section.
4. STUDIES OF DCA CARCINOGENICITY
4.1. HUMAN DATA
There are no epidemiologic studies available that assess carcinogenic outcomes in people
who have been exposed to DCA. Human exposures to DCA occur via drinking water
chlorination by-product contamination (U.S. EPA, 1994) and from chemotherapeutic treatment
for lactic acidosis and other metabolic and cardiovascular disorders (Stacpoole, 1989; Stacpoole "
et al., 1983, 1992; Eichner et al., 1974). Reports of these exposures., however, do not give any
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Inhibition in vivo by DCA-glutathione intermediates
Cytosol
*6lutalhlonetfansfefaseZetla s.(a
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insight into DCA's potential for carcinogenic effects. The drinking water studies do not uniquely
identify DC A because simultaneous exposures to many chlorinated and brominated by-products
can occur. Also, populations given DC A as a therapeutic agent have not had follow-up for
carcinogenic effects.
4.2. DCA CARCINOGENICITY BIOASSAY DATA IN RATS AND MICE
DC A is associated with increased incidences of liver tumors in both rats and mice. In
several studies in which DCA has been administered in the drinking water for an appreciable
fraction of the life span at concentrations ranging from 0.05 to 5.0 g/L investigators have
observed an increase in the incidence and multiplicity of hepatocellular adenomas and
carcinomas in both rodent species (Table 1). Results of these studies reveal that liver carcinomas
and preneoplastic lesions (adenomas, hyperplastic nodules, and altered foci) have been induced
by the highest concentration tested (typically 2 g/L to 5 g/L), but in two studies (Daniel et al.,
1992; DeAngelo, 1991), tumors were induced at 0.5 g/L . Preliminary data from an ongoing
study indicate increased tumor multiplicity may occur at 0.05 g/L (DeAngelo et al., 1998). The
tumor incidences reported in published studies range from 20% to 100%, and the multiplicity of
adenomas and carcinomas in several mouse studies is about 4, while in the rat studies
multiplicity is approximately 0.3. Concentrations down to 0.5 g/L for a lifetime have induced
tumors in 75% of the animals. A statistically significant elevation of either carcinomas alone or
adenomas counted together with carcinomas was seen in:
1. All six of the male B6C3F, mouse studies (Herren-Freund et al., 1987; Bull et al.,
1990; DeAngelo, 1991; DeAngelo et al., 1991; Daniel et al., 1992; Ferreira-Gonzalez
etal., 1995).'
-m
2. All but the last-cited of the following four female mouse studies: DeAngelo, 1991;
Pereira and Phelps, 1996; Pereira, 1996; Bull et al., 1990. The one report on female
mice where no response was detected (Bull et al., 1990) is not inconsistent with the
other reports because, with only 10 animals per group, for a duration of only 1 year, the
t; study had limited power to detect an effect.
3. Both of the two F344 rat studies (Richmond et al., 1995; DeAngelo et al., 1996).
The liver tumors have been observed in six published less-than-lifetime studies. In the mouse
study by DeAngelo et al. (1991), the time to the appearance, of the first tumor was significantly
shortened in the higher dose groups.
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Table 1. Animal cancer bioassays for dichloroacetic acid
Reference
Herren-
Freund et al.
(1987)
Species,
sex
B6C3F, -
Mice,
Male
Dose
Concentra-
tion (g/L)
0
ENU*
ENU + 2.0
ENU + 5.0
5-0
Dose rate
(rag/kg-day)
0
400
1,000
1,000
Water intake
(mL/kg-day)
200
200
200
Exposure
duration
(weeks)
61
61
61
61
61
Body weight
decrease
None
10%b
10%c
Necrosis
—
...
—
—
Incidence (percent of animals), |multiplicily|
Nodules
—
—
—
—
—
Adenomas
2/22
(9.1%)
2/22
(9.1%)
22/29
(76%)b
[1.4 bj
31/32
(99%)b
[5.3 b]
25/26
(96%)"
[4.6 "]
Carcino-
mas
0/22
19/29
(66%)"
[1.2-1
25/32
(78%)b
[1.5 "]
21/26
(8l%)b
[l.7b]
Total
tumors
...
...
...
...
...
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Table 1. Animal cancer bioassays for dichloroacetic acid (continued)
Reference
Bull et al.
(1990)
Bull et al.
(1990)
Species;
sex
B6C3F,
Mice,
Female
Male
Dose
Concentra-
tion (g/L)
0
2.0
0
1.0
2.0
2.0
Dose rate
(mg/kg-day)
170
Water intake
(mL/kg-day)
Exposure
duration
(weeks)
52
52
52
52
37+recovery
52
Body weight
decrease
—
None
None
None
Necrosis
—
—
...
Yes
Yes
Yes
Incidence (percent of animals), (multiplicity)
Nodules
—
3/10
(30%)
1/2
1/1
6/7
(86%)
9/10
i?o%i
Adenomas
—
—
0/1
0/1
2/7
(18%)
2/10
(20%)
Carcino-
mas
—
...
0/1
0/1
0/7
5/10
(50%)
Total
tumors
...
-
...
[0]
[0.25]
[2.0]
[4.0]
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Table 1. Animal cancer bioassays for dichloroacetic acid (continued)
Reference
DeAngelo et
al. (1991)
Species,
sex
B6C3F,
Mice,
Male
Dose
Concentra-
tion (g/L)
0
0.05
0.5
3.7
'
5.0
Dose rate
(mg/kg-day)
0
7.6
77.
410
486
Water intake
(mL/kg-day)
160
150
140
110
90
Exposure
duration
(weeks)
75
75
75
60
60
-
Body weight
decrease
None
None
I3%k
17%"
Necrosis
...
—
—
—
Incidence (percent of animals), (multiplicity)
Nodules
0/28
1/29
•
0/30
(58%)d
(83%)d
Adenomas
0/28
2/29 (7%)
1/27(4%)
12/12
(I00%)d
24/30
(80%)d
Carcino-
mas
2/28 (7%)
6/29
(21%)
2/27 (7%)
8/12
(67%)d
25/30
(83%)d
Total
tumors
A I O7%
[0.07]
AHC=
24%
A-iC=
11%
[0.11]
— .
[4.0]
...
[4.5]
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Table 1. Animal cancer bioassays for dichloroacctic acid (continued)
Reference
DeAngelo
(1991)
DeAngelo
(1991)
Daniel et
al. (1992)
Species,
Sex
B6C3F,
Mice,
Male
B6C3F,
Mice,
Female
B6C3F,
Mice,
Male
Dose
Concentra-
tion (g/L)
0
0.5
3.5
0
0.5
3.5
0
0.5
Dose rate
(mg/kg-day)
0
70
392
—
...
—
0
88
Water intake
(mL/kg-day)
—
140
112
—
...
—
197
190
Exposure
duration
(weeks)
104
104
104
104
104
104
104
104
Body weight
decrease
' *"*~
—
...
—
Necrosis
—
___
—
—
...
...
1/3 had
mild
necrosis
Incidence (percent of animals), (multiplicity)
Nodules
—
-— — .
—
—
...
—
0/20
2/24
(8%)
Adenomas
Carcino-
mas
A+C=I5%, [0.25]
A+C=75%, [1.4]
A+C=100%, [7.1]
A+C=8%, [O.I]
A+C=20%, [0.2]
A+C= 100%, [8.4]
1/20
(5%) .
10/24
(42%)c
2/20
(10%)
15/24
(63%)c
Total
tumors
3/20
(15%)
[0.25]
18/24
(75%)c
[1.4]
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Table 1. Animal cancer bioassays for dichloroacetic acid (continued)
K)
Reference
Richmond
et al.
(1995)
Ferreira-
Gonzalez
et al.
(1995)
Species, ,
Sex
F344
Rats,
Male
B6C3F,
Mice,
Male
Dose
Concentra-
tion (g/L)
0
0.05
0.5
2.4
0
1.0
3.5
Dose rate
(mg/kg-day)
0
—
—
—
—
—
—
Water intake
(mL/kg-day)
—
...
—
....
—
—
...
Exposure
duration
(weeks)
104
104
104
60
104
104
104
Body weight
decrease
—
—
—
'
—
— -
Necrosis
—
None
None
None
—
—
—
Incidence (percent of animals), (multiplicity!
Nodules
0/23
0/26
3/29
(10%)
19/27
(70%)
—
...
-^-
Adenomas
1/23 (4%)
0/26
6/29(21%)
7/27 (26%)
—
.—
—
Carcino-
mas
0/23
0/26
3/29
(10%)
1/27 (4%)
19%
[0.26]
71%
[1.3]
100%
[5.1]
Tolul
tumors
1/23
(4%)
0/26
12/29
(41%)
27/27
JJOO%)'
— .
...
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Table 1. Animal cancer bioassays for dicliloroacetic acid (continued)
Reference
DeAngelo
etal.
(1996)
Species,
Sex
F344
Rats,
Male
Dose
Concentra-
tion (g/L)
0
0.05
0.5
0
1.6
Dose rate
(mg/kg-day)
0
3.6
i.
40
0
139
Water intake
(mL/kg-day)
77
85
96
62
86
Exposure
duration
(weeks)
104
100
100
103
103
Body weight
decrease
None
None
27%
Necrosis
—
None
None
None
Incidence (percent of animals), [multiplicity |
Nodules
Adenomas
A=C=l/23
(4%),
[0.04]
A+C=0/26,
[-1
A+C=7/29
(24%)b,
[0.31]
A+C=l/33
(33%),
[0.03]
A+C=8/28
(29%)'.
[036]
Carcino-
mas
0/23
0/26
3/29
(10%)
1/33 (3%)
6/28
(21%)"
Total
tumors
—
—
—
—
—
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Table 1. Animal cancer bioassays for dichloroacetic acid (continued)
Reference
Pereira and
Phelps
(1996)
.
Pereira and
Phelps
(1996)
Species,
Sex
B6C3F,
Mice,
Female
B6C3F,
Mice,
Female
c-
Dose
Concentra-
tion (g/L)
MNUC + 0
MNU +
0.26
MNU +
0.86
MNU + 2.6
MNU + 2.6
0
2.6
Dose rate
(mg/kg-day)
...
—
...
—
—
—
Water intake
(mL/kg-day)
—
—
—
—
—
...
-™
Exposure
duration
(weeks)
52
52
52
52
11. then
recovery
52
52
Body weight
decrease
—
None
None
14%k
—
...
—
Necrosis
—
...
—
—
—
...
...
Incidence (percent of animals), (multiplicity)
Nodules
...
—
—
—
—
...
—
Adenomas
18%
[0.3]
20%
[0.2]
10%
[O.I]
73%
[3-6]'
46%
[0.7]
3%
[0.03]
35%
[0-45]
Carcino-
mas
10%
[0.1]
40%
[0.7]
20%
[0.2]
19%
[0.2]
15%
[0.15]
0
5%
[0.10]
Total
tumors
—
...
...
...
— .
...
...
-------
Table 1. Animal cancer bioassays for dichloroacetic acid (continued)
Reference
Pereira
(1996)
Species,
Sex
B6C3F, v
Mice,
Female
Dose
Concentra-
tion (g/L)
0
0.26
0.86
2.6
2.6, total
dose same
as 0.86
group
Dose rate
(mg/kg-day)
—
—
—
—
—
Water intake
(mL/kg-day)
90
50
28
19
34
Exposure
duration
(weeks)
82
82
82
82
82,
intermittent'
Body weight
decrease
None
None
17%"
—
Necrosis
—
—
—
—
...
Incidence (percent of animals), [multiplicity |
Nodules
—
—
—
...
—
Adenomas
2/90 (2%)
3/50 (6%)
[0.06]
7/28
(25%)'
[0.32 "]
16/19
(84%)"
(5.6 ']
3/34 (9%)
Carcino-
mas
2/90 (2%)
0
1/28(4%)
[0.04]
5/19
(26%)b
[0.37]
1/34 (3%)
0.03]
Total
tumors
...
—
—
...
'Ethylnitrosourea, as a single-dose initiator.
'Statistically significant compared to untreated controls, p < 0.05.
'Statistically significant compared to untreated controls, p < 0.01.
'Statistically significant compared to untreated controls, p < 0.001.
rN-methyl-N-nitrosourea as a single-dose initiator.
-------
In these assays there has been a consistent finding of increased liver weight (both absolute
and relative) shown to be primarily due to the enlargement of liver cells. The presence of cellular
necrosis that could lead to compensatory proliferation rarely has been observed (Stauber and
.Bull, 1997).
It is possible that the general health of the animals was impaired to such an extent by the
high doses that tumor -findings would not be relevant to low doses. Two measures of general
health were used: body weight and drinking water intake decrements relative to untreated
animals. Of the six studies that reported body weight changes, five of them (Herren-Freund et
al., 1987; DeAngelo et al., 1991, 1996; Pereira and Phelps, 1996; Pereira, 1996) reported body
weight decrements at the highest dose tested, which ranged between 10% and 27% of controls.
The remaining study (Bull et al., 1990) reported no body weight decrement. Of the six studies
where it was possible to evaluate drinking water intake as a function of DC A concentration, three
showed a decrement at the highest concentration (DeAngelo et al., 1991; DeAngelo 1991;
Pereira, 1996), the other three did not (Herren-Freund et al., 1987; Daniel et al., 1992, DeAngelo
et al., 1996). All seven of these studies had tumors at the high concentrations. From this
tabulation, EPA concludes that although it is possible that general health impairment, as
indicated by these measures, may contribute to .the tumor formation, this does,not seem to be a
requirement for the induction of tumors, even at the high doses. This conclusion does not agree
with that of the ILSI expert panel, which concluded that the animals were severely compromised
at the high concentrations (i.e., the maximum tolerated dose was exceeded) and that consequently
no statements can be made about tumor incidence at lower doses. Further discussion of the issue
appears in Section 6.
The limitations of the animal evidence are:
1. In only one study was a systematic histopathology evaluation of all tissues made; the
other studies examined only the liver. If more complete examination of tissues had
been done, sites in addition to the liver might have been discovered.
2. Many of the studies were done for less-than-lifetime durations at relatively high doses;
more information is needed at concentrations of 0.5 g/L and below with full lifetime
administration. This information would allow an evaluation of the lifetime effects of
lower concentrations.
3. The number of animals per dose group did not exceed 30 in any of the studies. For a
background incidence of 5%, as seen in the female mice and in the rat experiments, the
statistical power of the experiment is inadequate to detect less than about 33%
incidence in excess of the control incidence (Gart et al., 1986). This means that the
16
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low concentration could actually be causing as much as.33% excess incidence of
tumors in the animals and we would interpret the negative result as being without
effect. Therefore, if the animal experiments we.-e viewed as a model for the overall
population risk, they would be an inadequate safety screen. Note that the situation is
only slightly improved if the estimation is based on a standard bipassay of 50 animals
per group; here the estimation from a null result in animals could result in as much as
23% excess tumor incidence despite a negative bioassay result.)
Some or all of these rodent studies are summarized in greater detail in the review
evaluations mentioned in Section 2 above. It is significant to note here that DCA is a very
convincing hepatic tumorigen in rodents. Multiple tumors—as many as four per animal—have
been observed in a number of studies in which high doses of 2 g/L and above were administered
to male and/or female B6C3F[ mice. This effect occurred in less-than-lifetime studies with as
short a time as 1 year of treatment. Concentrations as low as 0.5 g/L have been observed to cause
a tumor incidence of about 80% in a lifetime bioassay (Daniel et al., 1992). DCA has also been
shown to cause liver tumors in male F344 rats when administered in drinking water (DeAngelo et
al., 1996; Richmond et al., 1995). Although peripheral neuropathy caused by high doses of DCA
occurred in the studies, evidence of liver tumors occurred at 60 weeks of treatment with 2.4 g/L,
and a tumor yield of 41% occurred in a group of 29 rats exposed to 0.5 g/L in their drinking
water for 2 years.
A preliminary report of a new study (DeAngelo et al., 1998) describes an increase in
rumor multiplicity in mice at a dose of 0.05 with no associated toxicity. In this same study,
tumors occurred in animals treated for only 10 weeks followed by 90 weeks maintenance. Stop
treatments of as early as 4 weeks are being carried out. This study in mice, as well as a National
Toxicology Program bioassay in rats and mice, will provide more definitive data for future
assessment of human risk from exposure to DCA.
/
4.2.1. Liver Tumors in Animals—EPA Guidelines Position
- The mouse hepatic neoplasia is the most common and controversial endpoint in the
rodent bioassays. The contentious disagreement surrounding the liver tumor response in the
male of the B6C3F, mouse strain specifically is well recognized. It is current EPA policy to
consider this endpoint relevant to humans unless chemical-specific data deem it otherwise (U.S.
EPA 1986, 1996), because it is the deregulation of molecular mechanisms that control such
processes as differentiation, proliferation, and death of cells that can be associated with cancer in
17
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all species, and therefore, tumor data from rodent carcinogenicity -bioassays are undoubtedly
useful in hazard identification.
The mouse liver target site usually is not a good predictor of carcinogenicity at the same
a
site in the rat, although increased mouse liver tumor incidence is a reasonably good predictor of
cancer at some site in the other species (Stevenson et al., 1990). In the case of DC A,
hepatocarcinogenicity is not confined to the mouse only, as liver tumors also develop rn rats
exposed to this chemical. Although the induction of tumors at a specific site in one species may
imply the induction of tumors at the same site in another species, this usually is not the case. Site
concordance among test species—as is the case with DCA regarding the finding of liver tumors
in both mice and rats—strengthens the weight of evidence in some respect. It is important to
recognize, however, that the induction of tumors in a specific organ site in test animals can be
said to imply the induction of tumors at any site in another species, including humans. Evidence
of tumorigenesis in a specific target organ in a rodent bioassay is, therefore, used to predict the
potential for human cancer hazard in general (U.S. EPA, 1986,1996)."
4.2.2. Sex Differences
It is interesting to note that there are differences in DCA tumor induction between sexes
m the B6C3F, mice. Tumors from DCA-treated female mice stained eosinophilic (Pereira,
1996), whereas tumors from male mice varied—smaller lesions stained 66% eosinophilic while
larger lesions were more basophilic (Stauber and Bull, 1997). Much shorter latency to tumor is
observed in male versus female mice. There are dissimilarities in the mutation frequencies
between tumors from male mice and tumors from female mice. Of tumors induced by DCA in
male B6C3F, mice, 50% to 60% exhibit mutations at codon 61 of the H-ras oncogene, not a
significantly different proportion from controls (Ferreira-Gonzalez et al., 1995; Anna et al.,
1994). This result differs from those obtained in female B6C3F, mice in which DCA reduced H-
ras codon 61 mutations in liver tumors to 4.5%, indicating that tumor formation is not associated
with a mutationally activated H-ras codon 61 (Schroeder et al., 1997).
5. MODES OF CARCINOGENIC ACTION THAT MAY RELATE TO LIVER TUMORS
There still exists a general relative lack of understanding of the pathogenesis of mouse
liver neoplasia; insight into the underlying molecular changes is limited at best. It is interesting
to note that various strains of mice can vary as much as 100-fold or more in susceptibility to
18
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chemically induced hepatocarcinogenesis. In the case of DC A administered to the B6C3F,
mouse, there is evidence supporting different modes of action for liver tumorigenesis, and even if
the compound is acting primarily as a tumor promoter, it may be acting through more than one
mechanistic pathway and that may vary with exposure level. Less information regarding mode of
carcinogenesis is available specifically for DCA rat liver tumor induction, although available
data are not inconsistent with related findings in mice.
5.1. MUTAGENICITY AND GENOTOXIC EFFECTS
Examination of the potential for DCA to produce genetic lesions such as gene mutations,
stable chromosomal aberrations, and aneuploidy could provide useful mechanistic information
for carcinogenesis given that genetic alterations are a component of the carcinogenic process.
Therefore, one of the first steps in understanding the possible mode of cancer induction for a
specific chemical is to conduct a thorough investigation of its potential mutagenicity. DCA has
been tested in vivo and in vitro for mutagenic potential and has not been clearly and consistently
shown to be an inducer of gene mutations or other types of genotoxicity. For example,
conflicting results have been reported regarding the ability of DCA to induce single-strand breaks
in hepatic DNA of rats and mice (Nelson and Bull, 1988; Nelson et al., 1989; Chang et al., 1992;
Daniel et al., 1993). Inconsistent findings among various reports of routinely used assays may be
attributed to artifacts or variations in conditions of acidity or purity of the test chemical.
Recently, a battery of standard mutageniciry tests was conducted at the EPA laboratory in
Research Triangle Park, North Carolina. These tests were designed and carried out to eliminate
or minimize shortcomings of some of the previous testing. They were intended to be a thorough
investigation of DCA mutagenicity. The results of this test battery were not all available when
previous reviews were performed by ILSI and EPA (ILSI, 1997; U.S. EPA, 1994; 1997; 1998
[actual review in 1996]).
5.1.1. Evidence for DCA Mutagenic Potential
The results of the more recent data available on the mutagenicity and genotoxicity of
DCA reported by Moore and her colleagues at EPA is consistent with the hypothesis that DCA is
a mutagen. Not all of these studies were available to the ILSI expert panel when they evaluated
the DCA database. The EPA studies consist of a basic evaluation battery that includes the
Salmonella bacterial assay (DeMarini et al., 1994), the in vitro mouse lymphoma gene mutation
assay (using the thymidine kinase locus) and gross chromosome aberration analysis, and the in
vivo analysis of the ability of the chemical to cause chromosomal damage (Harrington-Brock et
19
-------
al., 1998). The in vivo evaluation included both micronucieus induction in peripheral blood
erythrocytes and single-cell gel analysis of leukocytes—assays selected because of their
combined ability to detect and differentiate the general t> -pes of mutational damage that a
chemical may be capable of inducing (Fuscoe et al., 1996). In addition, to determine the ability
of DC A to induce gene mutations in vivo, the Big Blue Mouse Lac I assay was performed
(Leavitt et al., 1997). An evaluation was also conducted in the Microscreen Prophage-induction
assay (Fuscoe et al., 1996).
Strain TA 100 of Salmonella (using a vapor exposure) showed mutation induction both
with and without S9 (DeMarini et al., 1994). The lowest effective concentration was 50 ppm
(with S9). The mouse lymphoma assays evaluating both gene and chromosomal mutation and
gross chromosome aberration demonstrate the ability of DC A to induce primarily chromosomal
mutations in mammalian cells in vitro (Harrington-Brock et al., 1998). The concentration of
DCA required to see a response was relatively high compared with other carcinogens, but the
magnitude of the response was similar to that seen for "known genotoxic carcinogens." DCA
was active (with S9 exogenous activation) in the prophage assay, but only at very high
concentrations (Fuscoe et al., 1996). In fact, DCA is among the least potent of more than 100
chemicals evaluated in this assay. Studies.to evaluate the genotoxic potential of DCA in vivo
showed that it can induce a weak increase of micronuclei in peripheral blood erythrocytes and the
production of D.NA crosslinks in leukocytes (Fuscoe et al., 1996). An increase in Lac I mutants
also was seen for Big Blue transgenic mice exposed to DCA (Levitt et al., 1997). The in vivo
studies were done using doses similar to those used in the cancer bioassays.
To address the question of whether the slight increases in mutation observed in the
Salmonella assay and the Big Blue Mouse assay were due to DCA-induced mutation, and also to
identify the specific mutations that DCA induces, DNA sequence analysis was performed on the
Salmonella histidine revertants and the Lac I gene mutants. This sequencing analysis of the
Salmonella strain TA 100 histidine revertants found that DCA induced primarily GC > AT
transitions. Lac I mutants in DCA-treated animals showed a different mutational spectrum from
the control animals. This latter observation is particularly important because it argues that the
mutations were actually the result of newly induced mutations rather than an expansion of the
spontaneously preexisting mutants.
5.1.2. Information Regarding Mutation Spectra in DCA Induced Tumors
Several carcinogens have been shown to induce mouse liver tumors with specific point
mutations at codon 61 of the H-ras oncogene, a possible mechanism of cell transformation or
20
-------
initiation. DCA reduces H-ras codon 61 mutation frequency in liver tumors from female
B6C3F1 mice from about 50% to 4.5%, indicating that tumor formation is not associated with a
mutationally activated H-ras codon 61 (Schroeder et al., 1997). This result differs from those
obtained in DCA-treated male B6C3F1 mice which exhibited 50% to 60% of tumors with these
mutations, not significantly different from spontaneous tumors in controls (Ferreira-Gonzalez et
al., 1995; Anna et al., 1994). These dissimilarities between sexes are differences in mutation
frequencies. '
In a recent study,,Omer et al. (1998) observed somewhat lower mutation frequencies in
tumors from male mice treated with 0.5 and 2 g/L (lower doses than the 3.5'and 5 g/L
administered in the previous studies in male mice) when compared to spontaneous tumors. The
mutation frequencies reported by these investigators were 36% and 28%, respectively, for the
tumors from treated animals. In this study, mutation frequency was shown to increase with time,
although there was no consistent relationship with dose.
Information on mutational' spectra is potentially useful for distinguishing between
background and induced mutations. It should be noted that while,the mutation frequency is the
same for both the spontaneous tumors from control mice and the tumors from DCA-treated male
(but not female) mice, the mutation "spectrum for tumors produced by the high doses of DCA is
characterized by a lower percentage of AAA relative to CTA mutations when compared to
spontaneous tumors (Anna et al., 1994; Ferreira-Gonzalez et al., 1995; Orner et al., 1998).
This may suggest DCA causes selection against mutations giving rise to charged amino
acids corresponding to codon 61 in H-ras, but is neutral toward substitution of leucine for
glutamine, present in wild-type H-Ras protein. Structural differences due to charge may have
functional implications, possibly affecting binding affinity to proteins involved in signal
transduction (Drugan et al., 1996).
5.1.3. Summary
Results of an evaluation of genotoxic potential of DCA in a battery of short-term tests
conducted by EPA are consistent with its being mutagenic. The test results from the EPA battery
reveal the ability of DCA to cause mutational damage, although generally at relatively high
exposure levels. DCA induces both point mutations and chromosomal aberrations. The
induction of point mutations is usually considered to follow linear kinetics; however, other types
of genotoxic effects, particularly chromosomal aberrations, often do not occur with linear
kinetics.
21
-------
DC A has a metabolite (glyoxylate) that is mutagenic (Sayato et al., 1987; Sasaki and
Endo, 1978; Yamaguchi and Nakagawa, 1993; Mamett et al., 1985), although this metabolite is
also a chemical endogenous to the body, forming in transamination reactions involving glycine.
At least two potential reactive intermediates depicted in the DCA metabolic pathway are
discussed in Section 3. These DCA metabolites may be capable of binding to macromolecules
and possibly forming DNA adducts.
It should be mentioned that the ILSI expert panel report on DCA did not evaluate the
same genotoxicity data that EPA considered in this paper because not all of the studies had been
submitted for publication at the time of the ILSI panel meetings.
5.2. PEROXISOME PROLIFERATION
Chemically induced increases in numbers and/or size of hepatic peroxisomes, referred to
as peroxisome proliferation, has been suggested as the mode of action being related, in some
way, to the underlying mechanism through which some hepatocarcinogens cause liver cancer in
rodents. A class of nuclear receptors known as peroxisome proliferator-activated receptors
(PPARs) mediate at least some of the effects of certain hepatocarcinogens. PPAR-alpha is the
receptor mediating the effects of peroxisome proliferators in the rodent liver and is considered
key to initiation of cellular events leading to tumorigenesis, although the actual link between
receptor activation and development of tumors is unknown. xVast interspecies differences in the
expression of PPAR-alpha have been observed, particularly between humans and rodents, and
controversy exists regarding whether or not peroxisome proliferators are carcinogenic to humans.
DCA has been shown to be a weak peroxisome proliferator in mice (DeAngelo et al.,
1989; Daniel etal., 1992; Parrish et al., 1996). Mather etal. (1990) found increased activity of
cyanide-insensitive acyl CoA in rats, indicating that peroxisqme proliferation occurs, to some
extent, in this species as well since induction of the enzyme along with cell proliferation are
events associated with the overall process.
Elevations of c-Jun and c-Fos (Stauber and Bull, 1997) and increased expression of GST-
tt expression (Pereira and Phelps, 1996; Pereira et al., 1997) have been observed in experiments
with male and female B6C3F, mice, respectively. Elevations of c-Jun and c-Fos are expected to
increase GST-tt expression (Angel and Karin, 1991), whereas PPAR-alpha is known to hinder c-
Jun activity (Sakai et al., 1995) and thus, GST-it is not seen in peroxisome proliferator-induced
tumors. The observations that DCA tumors are immunoreactive to c-Jun and c-Fos, along with
other data showing expression of GST-ir in DCA-induced tumors, do not support peroxisome
proliferation as being a contributing mode of action to DCA tumorigenesis. In addition, single-
22
-------
strand breaks have been observed to result from DCA treatment before peroxisome proliferation
occurs (Nelson and Bull, 1988; Nelson et al., 1989), and DCA clearly produces tumors at doses
below those that are required for peroxisome proliferation (Richmond et al., 1995; DeAngelo et
al., 1989; Daniel et al., 1992; DeAngelo et al., 1996). And lastly, a higher H-ras mutation
frequency is observed in rumors induced by DCA than in tumors from mice treated with other
more potent peroxisome proliferating agents (Fereira-Gonzalez et al., 1995; Fox et al., 1990;
Omer et al., 1998; Anna et al., 1994). Taken together, these observations do not support a role
for peroxisome proliferation in contributing to DCA tumor induction.
5.3. EFFECTS ON THE INSULIN RECEPTOR
Smith et al. (1997) reported that DCA administration to mice at doses of 0.5 or 2.0 g/L
appreciably lowers their serum insulin concentration. The hypoglycemic effects of DCA have
been known for several years; in fact, DCA has been used therapeutically in people for this
outcome (Stacpoole et al., 1992; Stacpoole, 1998). Lingohr et al. (1998) have recently shown
that DCA modulates hepatocellular insulin receptor expression and signaling and that tumors
caused by DCA express elevated amounts of the insulin receptor. These investigators
hypothesize that certain tumor-promoting effects of DCA may be related to this DCA-induced
alteration of insulin signaling. Their findings of differences in the effects on initiated and normal
hepatocytes, including increased insulin receptor expression, increased Ras expression, and
increased MAPK phosphorylation in tumor cells compared with normal cells, are consistent with
the insulin-directed kinase cascade being active in DCA tumors. Insulin is known to have
mitogenic effects on the liver. The results of Bull and his coworkers imply that DCA can mimic
.insulin, alter the insulin signaling pathway, and provide a growth advantage to initiated cells
through a signal transduction pathway necessary for tumor growth (Kato-Weinstein et al., 1998).
It is possible, therefore, that the insulinlike effects of DCA could be involved in tumorigenesis.
DCA increases glycogen deposition in the liver similarly to insulin, and this response has been
observed in the same dose range as bioassay tumor induction.
5.4. ALTERATION IN DNA METHYLATION
Alteration in DNA methylation (5-methylcytosine content of DNA) is thought to play a
possible variety of roles in carcinogenesis (Jones, 1986; Holliday, 1987; Goodman and Counts,
1993; Goodman, 1997). Hypomethylation is considered to be a mode of action involved in
tumor promotion. Tao et al. (1998) have demonstrated that DCA-induced hypomethylation of
DNA in female B6C3F, mouse liver may be associated with promoting activity for this
23
-------
compound. The specific mechanism is not understood, but does appear to differ from
hypomethylation that is induced by a related compound, trichloroacetic acid, in the same study.
5.5. ALTERATIONS IN CELL REPLICATION AND DEATH RATES
Modification of cell replication and death rates may be an important effect of DCA
leading to tumor formation. The alterations may result in a mode of action based on suppression
and escape in which tumors arise from inititiated cells uninfluenced by DCA suppression of
mitosis. This mode of action is one that cannot be completely ruled out at lower doses. The
reported effects of DCA on cell replication are somewhat complex^. Increasing doses of DCA
increase the replication rates within initiated cells having a particular phenq type (Stauber and
Bull, 1997; Stauber et al., 1998). DCA administration to B6C3F, mice also appears to increase
the replication rates of normal hepatocytes in the short term; however, either chronic exposures
or high doses of DCA depress replication rates of these cells. Therefore, in normal cells, a
stimulatory effect is followed by a depression of replication rates. The inhibitory effect of DCA
on normal hepatocyte replication has been observed in different laboratories (Pereira, 1995;
Carter et al., 1995; Stauber and Bull, 1997). The higher the dose, the shorter the time to
inhibition. In contrast to normal cells, the hepatocytes within nodules and tumors are resistant to
DCA inhibitory effects (Stauber and Bull, 1997), and at higher doses there is a strong and
selective mitogenic effect of DCA on tumor cells that increases the growth rate of tumors with a
less malignant phenotype. This stimulus to growth rate may account for the nonlinear dose-
response relationship observed with DCA tumorigenesis, as well as progression of foci and
adenomas to carcinomas at high doses (Stauber and Bull, 1997).
Suppressed replication of normal hepatocytes in B6C3F, mice treated with DCA occurs
along with a decrease in apoptosis (Snyder et al., 1995). Both of these phenomena contribute to
suppressed cell turnover, which in turn may increase the probability of transformation of liver
cells and/or increased clonal expansion of damaged cells that would normally be extinguished.
The implications for risk assessment, however, may be different depending on which process is
involved, or whether both processes contribute to tumorigenesis. Downregulation of mitogenesis
may be somewhat less of a harmful process than depressed apoptosis with replication of cells
recognized as having DNA damage. Strong stimulation of tumor cell replication occurs at 2 g/L,
whereas selective suppression of normal hepatocyte replication, relative to initiated cells, likely
becomes more important to tumorigenesis at lower doses. Findings of Tsai and DeAngelo (1996)
indicate that this suppression is not due to an impaired ability of hepatocytes to respond to
growth factors.
24
-------
Elevated serum glucocorticoid levels have been shown to result from DCA treatment
(DeAngelo et al.)- Glucocorticoids influence a variety of functions, through binding with
intercellular receptors that are activated to bind to specific DNA response elements. Persistent
elevated glucocorticoid levels would give a continuous signal to activate/deactivate genes
involved in the control of cell replication and programmed cell death, which in turn, would
provide the opportunity for mutational or clonal selection events and the development of liver
cancer.
5.6. CYTOTOXICITY AND COMPENSATORY HYPERPLASIA
Normal cells can be converted to cancer cells by a multistage process. Every time a cell
divides there is a chance that a critical genetic error will occur, eventually leading to cancer
development. Thus, the likelihood of developing cancer can be increased when the number of
cell divisions in a critical target population of cells is increased. Regenerative hyperplasia in
response to cytotoxicity is one way that increased cell proliferation occurs. The ILSI expert
panel report relies heavily on the hypothesis that the tumorigenesis in the DCA rodent bioassays
is secondary to hepatotoxicity and associated necrosis that results in a proliferative response.
Under this view, the theory is that at low doses the cytotoxicity and necrosis would not occur,
and hence, neither would the tumors. As discussed below, there are some data to support tis
theory in mice, but not in rats, and other evidence is conflicting.
Liver cytotoxicity and necrosis is not observed following DCA exposure in rodents
except with longer treatments. Such effects are not reported in the rat carcinogenicity bioassays
(DeAngelo et al., 1996; Richmond et al., 1995) although Mather et al. (1990) reported a mild
increase in serum alanine aminotransferase in rats exposed to 5 g/L DCA in drinking water. (The
ILSI panel's personal communication with DeAngelo indicates toxicity is suggested by elevated
serum alanine aminotransferase levels in rats exposed to DCA [ILSI, 1997]. This is based on
findings in the Mather et al. [1990] study.) Evidence for increased lipoperoxidation in rats
treated with high doses was reported by Larson and Bull (1992a). Larson and Bull (1992a)
suggest that lipid peroxidation might be involved in DCA liver toxicity by having a role in
induction of the focal necrosis seen in B6C3F, mice (Bull et al.. 1990; Austin et al., 1996; Daniel
et al., 1992). Evidence for cytotoxicity and compensatory cellular regeneration in mice is
inconsistent among studies and at different dose levels. According to Sanchez and Bull (1990),
cytotoxicity is thought to be scattered individual cell necrosis or has been associated with
infarcted areas that are thought to result from severe cytomegaly.
25
-------
With prolonged DCA treatment, cytotoxicity and focal necrosis is evident at high doses in
some mouse studies. Such toxicity has not been morphologically documented in rats (DeAngelo
et al., 1996; Richmond et al., 1995; ILSI, 1997). This raises the question of whether tumor
formation is secondary to hepatotoxicity, at least in the rat. The finding of increased tumor
incidences without liver degeneration or with only mild evidence of hepatic effects, in both rats
and mice receiving 0.5 and 0.05 g/L DCA in drinking water, respectively, clearly does not
support that hypothesis (Richmond et al., 1995; DeAngelo et al., 1996; DeAngelo et al., 1998).
Conflicting opinions exist between some study investigators, risk assessors, and the ILSI expert
panel regarding whether or not there is ample evidence of hepatotoxicity and necrosis at all DCA
doses for cytotoxicity to be linked causally with liver cancer in the mouse studies. There is
clearly conflicting evidence regarding this issue with respect to disparities among labeling index
studies (Carter et al., 1995; Everhart et al., 1998; Sanchez and Bull, 1990; Stauber and Bull,
1997; Tsai and DeAngelo, 1996). This conflict is acknowledged in the ILSI report. Findings in
such studies clearly do not support the hyppthesis of tumors arising secondary to cell damage
followed by regenerative proliferation. Resistance to cytotoxicity observed in isolated
hepatocytes also does not support the hypothesis (Bruschi and Bull, 1993). In additon, data are
lacking to show that the degree of degeneration correlates with the excessive tumor incidence and
multiplicity observed in the various carcinogenicity assays. If these effects are truly important
forerunners to DCA hepatocarcinogenesis, the carcinogenic potency in rats would be
considerably less than what is observed. Necrosis does not occur in rats as a response to DCA
treatment; it is a finding unique to mice. In addition, necrosis must be clearly shown to precede
the development of tumors to eliminate the possibility that it is a simultaneous endpoint rather
than a precursor to tumorigenesis. This sequence has not been demonstrated.
No evidence of toxicity, particularly liver toxicity, is observed in patients administered
DCA doses of approximately 50 mg/kg/day (equivalent to a dose of 0.5 g/L in the drinking
water) for treatment of diabetes, hypeclipidemia, or lactic acidosis except for a marginal increase
in aspartate aminotransferase enzyme levels in a study of patients being treated for lactic acidosis
associated with severe malaria. No other liver enzymes were elevated (ILSI, 1997).
5.7. HEPATOMEGALY
Increased liver weight has been observed in both mice and rats as well as in dogs exposed
to DCA. Severe hepatomegaly in mice, thought to be due to increased cell size or cytomegaly,
was observed by Bull and his coworkers (Bull et al., 1990). Increased liver weight was observed
in B6C3.F, mice by Carter et al. (1995) with severe liver hypertrophy reported in the highest dose
26
-------
group: Mather et al. (1990) noted increased liver weight in rats exposed to DC A; however,
DeAngelo et al. (1996) did not observe this in their study of F344 rats chronically exposed to
DC A, although this may have been an artifact due to stuuy design. Increased glycogen was
discovered by PAS staining in both mouse hepatocytes (Bull et al., 1990) and rat hepatocytes
(Mather et al., 1990) following DC A exposure. Although increase in liver size may be due to
glycogen deposition, DCA-induced proliferative lesions were found to contain very little
glycogen relative to the surrounding hepatocytes (Stauber and Bull, 1997). Glycogen
accumulation does not occur following exposure to the DC A metabolites glycolate, oxalate, or
glyoxylate (Sanchez and Bull, 1990).
Hepatomegaly due to DCA exposure arises primarily from increases in cell size, although
it could also probably be associated to some extent with increase in cell number due to increased
replication rates. The hypothesis that a large amount of glycogen accumulation following DCA
exposure results in cell death and compensatory cell replication does not correlate well with the
findings of several investigators showing an inhibitory effect of DCA on normal hepatocyte
replication. Thus the findings reported by Carter et al. (1995) of increased liver weight at 0.5 g/L
with inhibition of cell replication, call into question whether cytotoxicity and reparative
hyperplasia is important as a contributing mode of DCA tumorigeneis. Similar results from other
studies (Pereira, 1995; Stauber and Bull, 1997) support these findings. Increase in liver size is
highly cofrelated with liver tumorigenesis in mice, however. A survey of carcinogenicity studies
on agrochemicals, for example, showed a clear correlation between hepatomegaly at 1 year and
liver cancer at the end of the studies (Carmichael et al., 1997). It is not yet understood whether
hepatomegaly caused by DCA exposure is related to tumor induction.
6. THE ISSUE OF DOSE LEVEL AND MTD
Historically, relatively high doses of test chemicals have been administered in chronic
rodent carcinogenicity bioassays to enhance sensitivity, thus increasing the ability to detect an
effect in small numbers of experimental animals. The use of high doses in cancer bioassays has
long been an area of scientific debate, largely because the possibility always exists that the mode
of carcinogenic action at high doses does not occur at tow doses, or it differs substantially from
mode of toxicity at low doses. Thus, the cancer-causing activity may occur only at the higher
doses and may not be relevant to human exposures to relatively lower doses.
In the case of dichloroacetic acid, the interpretation of bioassay results, in the context of
human hazard assessment, has become a contentious issue because the high doses administered
in the bioassays caused liver toxicity and necrosis in some mouse studies. Liver cytotoxicity was
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not observed in the rat bioassays, however (see Sections 4.2. and 5.6.). An important
consideration in the ILSI report was the hypothesis that dosing in the DCA bioassays exceeded
the MTD, resulting in liver toxicity that in turn led to carcinogenicity unlikely to occur below this
dose level, raising uncertainty about whether a carcinogenic response would occur at a lower
dose. Based on this view, the ILSI expert panel concluded that DCA cannot be classified
regarding its carcinogenicity in humans. Other concerns of various reviewers include the less-
than-lifetime studies, limited histopathology (in some studies, tissues normally inspected in
standard bioassays were not looked at), small numbers of animals, and whether health was
compromised due to decreased water consumption. The reduced study duration and small animal
group sizes in some studies decrease the study power to detect any response; yet, these
shortcomings did not preclude the induction of liver tumors in all but one case.
Dose selection for long-term studies is usually based on information from subchronic
studies and the MTD is a predicted value derived from observed toxicities in such studies. As
indicated in e-mail correspondence from A. DeAngelo, U.S. EPA to J. Parker, U.S. EPA dated
February 4, 1998, the MTD selected for the 1991 studies by DeAngelo and his colleagues
(DeAngelo et al., 1991, 1996; Daniel et al., 1992; Richmond et al., 1995) was either a dose that
resulted in a 10% inhibition of body weight gain when compared with controls, or, two log doses
below the 5 g/L high dose used in another study (Herren-Freund et al., 1987). The 10%
inhibition of body weight gain is within the limits designated in the EPA pesticide program
position document and the proposed EPA cancer guidelines (U.S. EPA, 1987,1996). Th«
highest dose was selected based on information from acute or prechronic studies. The high dose
in this study is similar to those administered to mice in other bioassay studies.
Whether the toxicity observed in the mouse bioassays is likely to be a causative mode of
action of carcinogeneis was discussed in Section 5 above. The degree of toxicity does not
correspond to the degree of tumor induction, the sequence of events is not demonstrated, and no
toxicity is observed in rats or mice at the lowest dose eliciting a tumor response (Richmond et al.,
1995; DeAngelo et al., 1996,1998). A pattern of toxicity, necrosis, and compensatory
proliferation was explicitly not occurring in the rat studies (DeAngelo et al., 1996; Richmond et
al., 1995). Sanchez and Bull (1990) concluded that aay cytotoxicity due to DCA exposure is in
-scattered individual cells or in infarcted cells resulting from cytomegaly. Findings of increased
liver weight along with inhibition of cell replication help to rule out cytomegaly as leading to cell
death and reparative hyperplasia as a mode of liver tumorigenesis.
The ILSI expert panel and other scientists have raised concern over the relevance of DCA
bioassay results to humans, and they do not believe that the mouse liver tumor response,
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particularly in the male B6C3F, mouse, is a relevant indicator of human cancer hazard. Much of
their concern relates to use of these data for quantitative low-dose risk extrapolation from high-
dose responses in rodents possibly associated with cytotoxicity precursor phenomena.
7. SUMMARY AND CONCLUSIONS
To ascertain the carcinogenic potential of a chemical, long-term chronic studies are
usually conducted in laboratory rodents. When administered to rodents in drinking water, DCA
is, beyond question, associated with aggressive hepatocarcinogenesis in male rats and both sexes
of mice, inducing multiple tumors—as many as four per animal—in a number of studies. Time-
to-tumor development is relatively short at the higher doses, and may decrease with increased
dose. Tumors have been observed to develop in animals exposed to DCA for only 10 weeks.
Concentrations as low as 0.5 g/L have been observed to cause a tumor incidence of about 75% in
a less-than-lifetime bioassay in mice, while concentrations of as low as 0.05 g/L have been
shown to cause an increase in tumor multiplicity with no evidence of toxicity. DCA has also
been shown to cause liver tumors in the absence of hepatotoxicity when administered to male
F344 rats in their drinking water. These observations are made in spite of the mostly
nontraditional bioassays.
Findings of cancer-causing activity in animals, such as that seen with DCA administration
raises concern for a potential human hazard. Reports of human exposures to DCA do not give
any insight into its potential for carcinogenic effects. The challenge then is to determine whether
the animal data, in this specific case, signify a potential human hazard.
The studies showing that DCA or its metabolites may react with DNA, resulting in DNA
damage and adducts, and subsequent mutations, are important to the assessment of DCA
carcinogenic hazard because such effects are highly likely to contribute to DCA's cancer-causing
activity. Particularly important are the results of an evaluation of the mutagenic potential of
DCA in a battery of short-term tests conducted at EPA's National Health and Environmental
Effects Research Laboratory (NHEERL). The findings from these studies on DCA genotoxicity,
with an emphasis on liver-specific endpoints, are consistent with its being mutagenic. This
evaluation reveals the ability of DCA to cause mutational damage, both point mutations and
chromosomal aberrations, although generally at relatively high exposure levels. Even so,
mutations are looked on as exhibiting linear low-dose responses according to EPA's Proposed
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1996), which regard a default
assumption of low-dose linearity as appropriate when the evidence "supports a mode of action of
gene mutation due to DNA reactivity." The DCA metabolite, glyoxylate, is also positive in the
29
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Ames assay. Whether the genotoxic responses to DCA, as well as to its metabolite, are relevant
to human hazard at expected DCA exposure levels in drinking water is not known; however,
direct DNA effects are generally thought to contribute to .the carcinogenic process, even at low .
doses. DCA mutagenicity findings are important; they are relevant across species, and, therefore,
cannot be dismissed.
Evidence is accumulating that supports a mode of action for DCA tumorigenesis through
modification of cell signaling systems, with preferential downregulation of control mechanisms
in normal cells without changing the proliferation rate of initiated cells, thus giving a growth
advantage to altered or initiated cells. Mitogenic proliferation of initiated cells, immune from
suppression of mitosis, may thus be an important factor in tumor development, although
contribution of cytotoxicity and compensatory proliferation at high doses and other mechanistic
contributions cannot be ruled out at this time. It is well accepted that carcinogenesis is a
multistage process; however, it is not clear what the actual roles of the several possible modes of
action for DCA tumorigenesis are. Additionally, there is very little basis for understanding
whether various possible mechanisms might contribute to DCA tumorigenesis at low doses,
although both mutagenesis and the suppression-escape modes of action are ones that are feasible
at lower doses. The data supporting particular modes of action do not imply species-specific
mechanisms, however. Until differences in the pathogenesis of DCA-induced tumors among
species can be identified, it is valid to acknowledge that there may be similarities in the process
across species. Although certain DCA effects are possibly unique to the high doses used in the
cancer bioassays, reasonable doubt exists that the mode of tumorigenesis is solely through
mechanisms that are operative only at high doses. Even so, DCA is likely to be carcinogenic in
humans at some dose because the existing evidence for modes of DCA carcinogenicity are not
species-specific. At low environmental exposures, greater uncertainty exists regarding the DCA
cancer hazard in humans.
DCA is clearly a rodent hepatocarcinogen, causing liver tumors in both rats and mice in
multiple studies; however, the modes of action through which it induces liver tumors remain
unclear. Several different events may be occurring in the liver following exposure to DCA, and
data exist to support, to some extent, completely different modes of tumor induction that are not
species-specific, including mutagenesis. The transspecies hepatocarcinogenic effects of DCA,
along with other interspecies effects related to possible modes of tumorigenesis, are adequate to
demonstrate a potential human hazard. The combined weight of experimental evidence suggests
that this chemical should be considered likely to be carcinogenic to humans. There is, of course,
a considerable degree of uncertainty concerning the likelihood of a human hazard associated with
30
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exposure to low levels of DC A usually encountered in the environment or in drinking water. The
fact that DCA inhibits its own metabolism may be important in evaluating effects at lower doses.
Also important to consider is the fact that, in the case of DCA, the overall accumulation of
bioassay evidence concerning dose requirements for rumor induction indicates the importance of
"concentration * time" or total dose to eancer-causing activity. Thus, the possibility of tumor
induction resulting from exposure to a lower dose over a much longer time period cannot be
eliminated. Again, it needs to be emphasized that mechanism(s) of DCA carcinogenesis are
simply not understood well enough to know.
Although DCA is deemed likely to cause cancer in humans at some.dose level, the shape
of the dose-response curve for tumor development may be quite complicated, and the slope of
this curve may vary appreciably depending on the dose range considered. It is not surprising that
the shape of the dose-response curve would be complex considering the many interactive
processes that could contribute to carcinogenesis. These processes, especially when taken
together over a wide range of doses, are highly unlikely to exhibit a linear dose-response
relationship. Mechanistic considerations may justify special interpretation of the dose-response
data with respect to calculating human cancer risk. It must be remembered that this report refers
only to the weight of the experimental evidence that DCA is carcinogenic and not to its potency
of carcinogenic action. This report has not addressed directly the quantitative estimation of risk.
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U.S. Environmental Protection Agency. (1994, March 31) Final draft for the Drinking Water Criteria Document on
chlorinated acids/aidehydes/ketones/alcohols. Prepared by Clement International Corp for Office of Water.
U.S. Environmental Protection Agency. (1996) Proposed guidelines for carcinogen risk assessment. Federal
Register 61:17960-18011. •
U.S. Environmental Protection Agency. (1997, Oct. 10) Summary of new health effects data on drinking water
disinfectants and disinfectant byproducts for the notice of data availability, [draft]. Prepared by Cadmus Group Inc.,
Waltham, MA, for the Office of Water, U.S. Environmental Protection Agency, Washigton, DC, under EPA
contract no. 68-C7-0002. .
.U.S. Environmental Protection Agency. (1998) Integrated Risk Information System (IRIS) file on DCA. Online.
National Center for Environmental Assessment, Washington, DC.
36
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Yamaguchi, T; Nakagawa, K. (1993) Mutagenicity of formation of oxygen radicals by trioses and glyoxal
derivatives. Agric Biol Chem 47:2461-2465.
37
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Appendix A: Charge to Peer Reviewers and Comments
Statement of Work
TITLE: Peer review of Health Risk Characterization Report on Dichloroacetic Acid
BACKGROUND: The mission of the United States Environmental Protection Agency's (EPA)
Office of Water (OW) is to protect public health and the environment from adverse effects of
contaminants in media such as ambient water, drinking water, waste water, sewage sludge and
sediments. This procurement relates to the peer review of a health risk assessment on the
chlorination disinfection by product, dichloroacetic acid. This risk assessment will be used in
support of EPA's stage 1 disinfection by product rule which is scheduled to be final in November
1998. The Safe Drinking Water Act Amendments of 1996 emphasize that "the best peer review
science" be used in carrying out SDWA regulations.
PURPOSE: A cancer hazard weight of the evidence characterization has been recently prepared
on dichloroacetic acid (DCA). This 1998 document considers a 1997 report prepared by the
International Life Sciences Institute,(ILSI), as well as new data published after the EPA 1994
assessment concerning the carcinogenic mode of action. This assessment does not present a
dose-response assessment for DCA because the current data are considered inadequate for risk
quantification. This assessment also applies the EPA's 1996 proposed revisions to its guidelines
for carcinogen risk assessment.
TASK DESCRIPTION: This purchase will procure a peer review on the 1998 EPA DCA
hazard characterization. EPA has attached 1998 the DCA report, consisting of 20-25 pages, to be
reviewed (Attachment 1), as well as supporting materials, such as EPA's 1996 guidelines for v
carcinogen assessment (Attachment 2), EPA's 1994 Criteria Document on DCA (Attachment 3),
and ILSI report (Attachment 4). The peer reviewer shall submit written comments that are
clear/transparent, and constructive. They shall comment on whether the document clearly and
adequately discusses:
- the weight of the evidence
- the key lines of evidence
- the mode of carcinogenic action understanding'
- uncertainties in the risk assessment
The peer reviewers shall indicate where they are in agreement with the report and where they
disagree. If they disagree with any part of the report or find a weakness in the report, they shall
recommend explicit guidance on revising the report. They shall provide comments that include
an overall general summary on the acceptability and adequacy of the hazard characterization, and
specific comments as needed (comment 1 on page X, paragraph X, line X).
A-l
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Comments on Dichloroacetic Acid:
Carcinogenicity Identification
Characterization Summary
Prepared by
R. Julian Preston, PhD
Chemical Industry Institute of Toxicology
6 Davis Drive
Research Triangle Park
NC 27709
USA
February 15,1998
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Introduction
This peer review of the document "Dichloroacetic Acid: Carcinogenicity Identification
Characterization Summary" provides a discussion of the key data that are utilized for
carcinogenicity assessment and the proposed mode of action underlying the carcinogenicity.
Additional information was obtained from the ILSI Report, An Evaluation of EPA's Proposed
Guidelines for Carcinogen Risk Assessment Using Chloroform and Dichloroacetate as Case
Studies (November, 1997). The nature of the data available on the carcinogenicity and
mutagenicity of dichloroacetic acid (DCA) dictates that the report be rather speculative. The
arguments are reasonably well-developed, although requiring some more in-depth discussion of
the quality of the data being considered in support of the carcinogenic potential of DCA and its
possible mode of action.
Weight of the Evidence '
There is no evidence for DCA-induced tumors in humans and, in fact, very little information
on potential lexicological responses. This clearly makes it very difficult to establish that data
obtained in rodent carcinogenicity studies can be used to predict effects in humans. Such an
extrapolation is further compounded by the fact that the carcinogenicity data from rodent studies
presents a number of problems as outlined and generally appropriately discussed in the present
report, as well very precisely in the ILSI Report of an Expert Panel (November, 1977). More
recent studies (DeAngelo et al., 1996 and Richmond et al., 1995) provide somewhat more reliable
evidence for hepatocarcinogenicity in male F344 rats and B6C3F1 mice, but as noted there are
complications to the interpretation based upon experimental design and dose selection. The most
recent study of DeAngelo et al (1998) is not available for review and so was not considered in
the present peer review to provide supporting evidence for carcinogenicity. As a general
conclusion, it seems likely that DCA exposure can result in liver tumors in rodents, particularly at
high concentrations, but the specific nature of this induction remains unclear. The utility of the data
is unclear as discussed in the report and the ILSI Expert Panel Report, since liver tumors in
B6C3F1 mice remain a controversial endpoint for predicting effects in humans. The data for
carcinogenicity in rats are not sufficiently sound to allay this concern by DCA being a two-
species carcinogen.
The mutagenicity and genotoxicity data remain somewhat equivocal. The report needs to
be somewhat more critical in its discussion of the mutagenicity data. The conclusions drawn on
the mutagenicity of DCA are not as clear cut as described. Thus, their inclusion into a mode of
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action is not particularly well grounded. Reference to the ILSI Report of the Expert Panel is
appropriate, since their discussion is clear and precise. The information on mutational spectra is
potentially very useful for distinguishing between background and induced mutations. Additional
supportive data are needed, however.
The weight of evidence presented for other potential modes of action is relatively weak. A
proposed mode of action proposes that OCA in humans produces peroxisome proliferation and
subsequent cell proliferation. There is some more recent data to support that DCA functions
through PPAR, but whether this affects proliferation has not been established. Similarly, the data
to support the suggestions that DCA can alter cell replication and death rates and/or induce
cytotoxicity and compensatory hyperplasia that can subsequently lead to tumor formation are
preliminary at best. The discussions in the report are generally reasonable and the conclusions
reached are appropriate. *
Mode of Action for Carcinogenicitv
The mode of action discussion in the report, under the section headed Summary and
Conclusions (pages 22-24) is generally well balanced and fair. The argument follows the
generally accepted paradigm that cancer is a multi-step process and that mutations determine
passage through the various steps. Cell proliferation is necessary for the process as a mutation
effector or for the amplification of preexisting individual cells. However, the evidence for
mutagenicity, mitogenicity and/or cytotoxicity and regenerative cell proliferation is quite weak at
this time. This is especially true if effects at low exposure levels are being considered. The lack of
information does not allow for a dose response characterization, based upon a reliable mode of
action, to be developed. The places where additional information will have an impact are
abundantly clear, and until this time any mode of action discussion is premature.
Uncertainties in the Risk Assessment
The uncertainties basically fall out of the above discussions. The carcinogenicity of DCA
in rodents has to be more firmly established by a 2-year bioassay designed for this purpose.
Utilizing Ijver tumor data from B6C3F1 mice remains equivocal, making reliable tumor data for the
rat a necessity. The lack of any very informative data on DCA effects in humans makes
extrapolation from data in laboratory animals to humans open to concern.
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There are dearly uncertainties in the mode of action for providing information pertinent to
tumor dose response characterization. While point mutations are predicted to be linear with dose,
the tumor dose response curve even when mutagenicity is implicated, is a reflection of the
probability of mutation induction as a consequence of other cellular perturbations. These latter
events are more than likely non linear with dose. Thus, predicting the shape of this dose
response curve for DCA-induced tumors, (presuming they can be induced) requires knowledge
beyond what is currently available. As a side note, mutagenicity does not dictate linearity for
tumor dose response curves at low doses. However, a knowledge of mode of action is needed to
support non linearity'. The current data for DCA are not able to provide this.
Conclusions
The report on the potential carcinogenicity of DCA is perhaps^pss critical than it needs to
be for establishing hazard identification (tumorigenicity and mutagenicity). The discussions on
mode of action, on the other hand, are clear and rational. The bottom line that "DCA is deemed
likely to cause cancer in humans at some dose levels" would seem to be premature at this time.
As noted in the weight of evidence discussion above, a firm conclusion of human carcinogenicity
is not warranted by the data.
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Lauren Zeise
Comments on Dichloroacetic Acid
Carcinogenicity Identification Characterization Summary
March 1998
Overall the document is well written and is adequate for the purpose of providing a
hazard characterization for the carcinogenicity of dichloroacetic acid in humans. The
brief format works well; the level of detail is sufficient to enable the reader to understand
US EPA's interpretation of the data and its position regarding weight of evidence. The
weight of evidence finding is adequately supported by the analysis provided. Key lines of
evidence are adequately discussed. The section on mode of action is particularly well
done. The uncertainties in the hazard characterization call are discussed at length. Issues
raised by the ILSI report are adequately addressed.
Other general comments are mixed with editorial comments below. Given the short time
frame for revision of the document there may not be enough time to make many editorial
changes. Nonetheless, the document as is meets the objectives identified and contains no
major flaws. The one technical addition to the document that would be most helpful
would be the inclusion of values of statistical significance (including trend tests if
possible) to the tabulation of animal studies.
Specific Comments
Page 1, line 24. The statement that a hazard characterization is only provided because
"the available published studies are not considered adequate to support biologically based
quantitative dose-response estimation" begs the question of whether there is enough
quantitative information to provide numerical guidance levels. The discussion on page
24 suggests that there may be.
Page 2, line 15, LARC needs to be identified (i.e., as the International Agency for
Research on Cancer).
Page 2, line 25. It would be helpful to inform the reader of lARC's finding regarding
animal evidence. .
Pages 6 and 11, in the discussion of study limitations it would be useful to include for
each noted limitation a sentence on the implications for the weight of evidence
determination. For example, regarding the first noted limitation, one cannot determine
the impact of DCA on the carcinogenesis at sites other than the liver. The statement that
the bioassay duration was less than "the acceptable standard for rodent studies" may be
subject to misinterpretation by the general reader. Perhaps noting that this results in a
reduction in study power would help. Similarly under the fourth noted limitation, in
making statements regarding the adequacy of the study due to the numbers of animals the
implications regarding study power could also be discussed.
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Comments on DCA
Pages 7-10. It would be helpful to add values of statistical significance (including trend
tests if possible) to the tabulation of animal studies and an indication of which cells are
left empty because the study report did not provide the information.
Page 11, lines 28-29. The statement regarding the liver tumor controversy overstates the
case somewhat. While the findings of hepatic tumors in B6C3F1 male mice may be more
difficult to interpret regarding implications for humans, findings in the female of this
strain and in rats are less so (i.e., no more so than for most other rodent sites).
Page 12, line 1. Suggest confining the statement to male 86C3F1 mice.
Page 12 discussion of site concordance. While the mouse liver may not be a good
predictor of whether or not liver tumors will be induced in rats, it is a reasonably good
predictor that cancer will be increased at some site in another species.
Pages 13-20. The section on mode of action is particularly well done and provides the
background necessary to understand the Agency's position.
Page 16, line 29. Nelson and Bull 1988? (year missing in citation)
Page 21, line 3. Liver toxicity and necrosis was seen at some but not at all dose levels
associated with liver carcinogenicity.
Page 21, lines 8-11. The concerns raised should be addressed with respect to liver tumor
induction. For example, is there any evidence that reduced water consumption would
cause liver tumors in humans? Also, the reduced study duration reduces study power, yet
this was not sufficient to preclude the induction of liver cancer in all but one case.
Page 23. It would be helpful to let the reader know whether, under the old guidelines, the
Agency would have revised its position.
Page 24, line 2. Use of the word "completely" seems to overstate the case.
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Comments on DCA Carcinogenicity Characterization
James A. Swenberg, D.V.M., Ph.D.
University of North Carolina
Chapel Hill, NC 27599
919-966-6139 Tel
919-966-6123 FAX
James_Swenberg@unc.edu
The draft carcinpgenicity characterization of DCA provides a reasonable overview of
most aspects of the pertinent data on the chemical. However, the characterization needs to be '
enhanced by providing more information on the quantitative nature of some of the data. In
particular, tables that provide the doses and responses of the genetic toxicity data are needed.
There is a major discrepancy between the interpretation of the genotoxicity data in this document
and in the ELSI review of DCA. It is therefore imperative that these differences be shown in a
transparent manner. Were the positive results in the same dose range as the carcinogenicity data
for in vivo studies and were the concentrations used in the in vitro studies similar to metabolite
studies in animals? Likewise, data on metabolism needs to be placed in perspective to the doses
used and exposure duration. Do pathways saturate? Underwhat conditions? The general lack of
quantitative relationships detracts from the document.
It is difficult to understand why we have such an unusual data base from the standpoint of
standardized protocols and tissue evaluations for this chemical. I recognize that this is not the
fault of the carcinogenicity characterization. Never-the-less, it decreases the utility .of these data.
Some issues could still be addressed by additional studies on the available.materials. For
example, one or more Pathology Working Groups could be set up to evaluate hepatotoxicity
using standardized criteria and blinded scoring of the slides. This is really necessary, as much of
the data appears to have been generated by scientists that are not board certified pathologists and
no information on blinding of the pathology reading was provided. The ELSI review panel did
review the slides of some of the studies, but a report is not available. A data package like that
available on DCA would not be accepted by the EPA for registration of a pesticide.
The identification of hepatic tumors after as little as 10 weeks of exposure to high doses
increases the level of concern for this chemical.. The data from Daniel et al.,' 1992, should be
added to Table 1. The first line of Bull et al., 1990, should show 0/10 for hepatic tumors if that is
the case. It is now blank. Likewise, the DeAngelo et al., 1991, 3.5 g/L carcinoma data should be
67%, not 6%.
The carcinogenicity characterization discusses DCA's mutagenicity in several places,
with comments that mutagenic events are expected to be linear. This is stretching the common
default. The default is that mutagenic processes are expected to have low dose linearity when
none of the processes are saturated or depleted. That is not the same as saying that genotoxic
events associated with high exposures to DCA are linearly related to such events at low doses,
since there is clear evidence for nonlinear responses at the doses evaluated. If oxidative stress is
involved at high doses, it in unlikely that similar processes occur at much lower doses. Thus, I
thought that the arguments for linearity were weak. When this is coupled with the marked
difference in opinion on the interpretation of.these assays between the ILSI document and this
document, it is "clear that a better and more transparent process is needed to present the data and
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conclusions.
Many of the references cited are only abstracts. Others represent unpublished reports.
Such documents leave much to be desired from the standpoint of being available for peer review.
Some mechanism should be provided to make these documents accessible to interested parties.
A review of the amounts of DC A present in drinking water and the potential for human exposure
would also be helpful.
In summary, by providing additional data to the report, it will be improved. This report
highlights the need for high quality data for mode of action decisions. It is unusual in that mere
is considerable mechanistic data, but has a less than acceptable toxicology data package. Even
with all the mechanistic data, however, a clear mode of action is not evident. There is little doubt
that hepatotoxicity occurs at the doses causing liver cancer. The. fact that sustained increases in
cell proliferation are not maintained weakens this evidence. The genotoxicity is clearly
controversial and may very well be due to secondary mechanisms associated with oxidative
stress and its associated lipid peroxidation. As such, it would not be expected to be linear. There
are data available on human responses that suggest that humans may be less susceptible to
hepatotoxicity. I agree that only a crude estimate of risk can be made at this time. In my
opinion, this should be presented as both linear and nonlinear approaches, with most arguments
supporting a nonlinear approach. I am not at all convinced that the genotoxicity data are
consistent with a linear mechanism
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Appendix B: Remarks on Peer-Review Comments
EPA received a number of comments and suggestions to the External Review Draft of the
DC A paper (February 1998) from both Agency scientific staff and external reviewers. Written
comments from three expert external peer reviewers are included in Appendix A. The majority
of their remarks were mostly favorable. Generally, the paper is said to be well-written and the
discussion of mode of action is very good. The reviewers raised several points about some of the
more controversial issues, and they identified a number of specific statements that needed to be
clarified or changed to be entirely accurate. In some cases, the external reviewers do not agree
with each other, and the comments are somewhat conflicting.
The recommendations of all of the reviewers were considered, and they have been
incorporated into the manuscript where appropriate and possible during the short turnaround
time. The EPA greatly appreciates these helpful suggestions and believes the paper is improved
by clearing up certain points and by including additional information provided by commenters.
A major concern of two of the three external reviewers and some of the internal reviewers
was why EPA's conclusions differ from those of the ILSI expert panel's "using the same data"
and, particularly, why EPA differs from ILSI and others in the interpretation of the mutagenicity-
related data. It is important to point out that the ILSI expert panel was convened in 1996, and
their final report came out in 1997, whereas the EPA hazard characterization was prepared in
early 1998. Thus, the database reviewed was not the same, and it continues to change even now.
EPA reviewed a different database that included crucial studies not considered by the ILSI expert
panel. This difference in the data reviewed is also the case regarding the
mutagenicity/genotoxicity studies specifically. Not all of the studies from the complete test
battery conducted by the EPA's own National Health and Environmental Effects Research
Laboratory were published and available to the ILSI expert panel. Consideration of the complete
test battery makes a difference in the EPA's interpretation of potential DCA mutagenicity. The
remaining studies from the test battery conducted by EPA have now been accepted for
publication in peer-reviewed scientific journals and are in press. Taken as a whole, this test
battery indicates that DCA is mutagenic at the doses of the bioassays, and a role for mutagenicity
as a contributing mode of tumorigenicity cannot be ruled out for low doses, although it may not
be as prominent a factor as it is at the higher dose levels due to interplay with other events that
are dose-related.. On the other hand, mutagenicity may have a relatively more predominant role
in tumorigenesis at lower doses because certain other factors are not yet operative at these doses.
B-l
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The overall dose-response curve is probably complex, with low-dose cancer potency not likely to
be directly extrapolatable from high-dose potency.
Another key issue identified by the external peer reviewers is the question of the linearity
of the dose-response curve. Although DC A is deemed likely to cause cancer in humans at some
dose level, the shape of the dose-response curve for tumor development may be quite
complicated, and the slope of this curve may vary appreciably depending upon the dose range
considered. It is not surprising that the shape of the dose-response curve would be complex
considering the many interactive processes that could contribute to carcinogenesis. These
processes, especially when taken together over a wide range of doses, are highly unlikely to
exhibit a linear dose-response relationship. In fact, each of the individual processes is most
likely to be nonlinear. Even so, mutations are looked upon as exhibiting linear low-dose
responses according to EPA's Proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA,
1997) which regard a default assumption of low-dose linearity as appropriate when the evidence
"supports a mode of action of gene mutation due to DNA reactivity."
It must be remembered that this report refers only to the weight of the experimental
evidence that DC A is carcinogenic and not to its potency of carcinogenic action. This report has
not directly addressed the quantitative estimation of risk. Mechanistic considerations of DC A
tumorigenesis may justify special interpretation of the dose-response data with respect to
calculating human cancer risk.
B-2
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Parker, August, 1998
Re: DCA Comments
Response to Comments
on
Dichloroacetate
Peer Review
The National Center for Environmental Assessment-Washington Office prepared a paper
issued in March, 1998, entitled: " Dichloroacetic Acid: Carcinogenicity Identification
Characterization Summary". The February External Review Draft of this report was reviewed in
accordance with U.S. £PA policy and approved for publication. Both Agency scientific staff and
official external reviewers made comments and suggestions on the review draft.
All comments considered
All of the recommendations of the reviewers were considered, and these were
incorporated into the manuscript where appropriate. Many of the suggestions from the external
peer reviewers were especially helpful in revising the February draft of the paper, making the
final March report an improved document. Certain important points were clarified in the final
report based upon the suggestions made by peer reviewers. Also, the final report included
additional relevant and important information that had not been discussed in the review draft.
Some of this information was provided by commenters, including peer reviewers .
Comments generally favorable
The written comments from the three external expert peer reviewers were included in
Appendix A of the published DCA manuscript. The majority of the remarks were mostly
favorable overall. Generally the paper was said to be well-written and the discussion of mode of
action very good. For example, one peer reviewer stated that the document "is well written" and
that it "is adequate for the purpose of providing a hazard characterization for the carcinogenicity
of dichloroacetic acid in humans," that the "brief format works well," that the "level of detail is
sufficient to enable the reader to understand US EPA's interpretation of the data and its position
regarding weight of evidence," that the "weight of evidence finding is adequately supported,"
that "key lines of evidence are adequately discussed," and that "the section on mode of action is
particularly well done." This reviewer also noted that the "uncertainties in the hazard
characterization are discussed at length" and that" the issues raised by the ILSI report are
adequately addressed."
Commenters do not always agree with each other
The reviewers raised points about the more controversial issues, and they identified a
number of specific statements that needed to be clarified or changed to be entirely accurate. In
some cases, the external peer reviewers do not agree with each other, and their comments are
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Parker, August, 1998
Re: DCA Comments
somewhat conflicting.
Important concern of two of three peer reviewers
A major concern of two of the three external reviewers and some of the internal reviewers
was why EPA's conclusions differ from the ILSI expert panel's conclusion "using the same
data"and, particularly, why EPA differs from ILSI and others in the interpretation of the
mutagenicity-related data. It is important to point out that the ILSI expert panel was convened in
1996 and their final report came out in 1997, whereas the EPA hazard characterization was
prepared in early 1998, Thus, the database reviewed was not the same, and it continues to
change even now. EPA. reviewed a different database .that included crucial studies not
considered by the ILSI expert panel. This difference in the data reviewed is also the case
regarding the mutagenicity/genotoxicity studies specifically. Not all of the studies from the
complete test battery conducted by the EPA's own National Health and Environmental Effects
Research Laboratory were published and available to the ILSI expert panel. Consideration of the
complete test battery makes a difference in the EPA's interpretation of potential DCA
mutagenicity. The remaining studies from the test battery conducted by EPA have now been
accepted for publication in peer-reviewed scientific journals and are in press. Taken as a whole,
this test battery indicates that DCA is mutagenic at the doses of the bioassays, and a role for
mutagenicity as a contributing mode of tumorigenicity cannot be ruled out for low doses,
although it may not be as prominent a factor as it is at the higher dose levels due to interplay with
other events that are dose-related. On the other hand, mutagenicity may have a relatively more
predominant role in tumorigenesis at lower doses because certain other contributing factors are
not yet operative at these doses. The overall dose-response curve is probably complex, with low-
dose cancer potency not likely to be directly extrapolatable from high dose potency.
Peer reviewers comment about dose-response curve linearity
Another key issue identified by external reviewers is the question of the linearity of the
dose-response curve. Although DCA is deemed likely to cause cancer in humans at some dose
level, the shape of the dose-response curve for rumor development may be quite complicated, and
the slope of this curve may vary appreciably depending upon the dose range considered. It is not
surprising that the shape of the dose-response curve would be complex considering the many
interactive processes that could contribute to carcinogenesis. These processes, especially when
taken together over a wide range of doses, are highly unlikely to exhibit a linear dose-response
relationship. In fact, each of the individual processes is most likely to be nonlinear. Even so,
mutations are looked upon as exhibiting linear low-dose responses according to EPA's Proposed
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1997) which regard a default assumption
of low-dose linearity as appropriate when the evidence "supports a mode of action of gene
mutation due to DNA reactivity."
One peer reviewer stated that "only a crude estimate of risk can be made at this time." and
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Parker, August, 1998
Re: DC A Comments
suggested that both linear and nonliear approaches should be presented. Another peer reviewer
states that the discussion in the paper suggests there may be enough evidence to support
biologically-based quantitative dose-response estimation. It must be remembered that this DCA
paper refers only to the weight of the experimental evidence that DCA is carcinogenic and not to
its potency of carcinogenic action. This specific report has not directly addressed the quantitative
estimation of risk. The paper does state that mechanistic considerations of DCA tumorigenesis
may justify special interpretation of the dose-response data with respect to calculating human
cancer risk.
Quality of data, uncertainty, and an all important basic difference in thinking
Peer review comments included those of one of the three reviewers that "the iarguments
are reasonably well-developed, although requiring some more in-depth discussion of the quality
of the data being considered in support of the carcinogenic potential of DCA." This paper is a
summary carcinogenicity identification characterization. It is not meant to be a detailed, in-depth
discussion of all data. It is, however, considerably more comprehensive than the "two to five
page summary" originally requested, because of the amount of information which had not been
previously reviewed, as well as the controversial and conflicting nature of some of the data.
Again, this paper is a summary and is not meant to stand entirely alone in presentation of
particulars. The details of studies are found elsewhere.
As is pointed out by peer reviewers, the data quality is clearly an issue. EPA agrees, and
this is a main reason why the paper concludes that a "considerable degree of uncertainty" exists
regarding "the likelihood of a human hazard associated with exposures to low levels of DCA
usually encountered in the environment or in drinking water". It appears that the primary
difference in thinking between one reviewer's comments and the EPA paper is in the
interpretation of the direction to take because of the uncertainty involved. EPA agrees with the
reviewer that some of the data are difficult to interpret. The reviewer states that the "mode of
action discussion in the report.....is generally well balanced and fair." EPA agrees with this
reviewer that "a firm conclusion of human carcinogenicity is not warrented at this time," but does
not agree with this reviewer that the "bottom line" of the EPA paper that "DCA is deemed likely
to cause cancer in humans at some dose levels" "would seem to be premature at this time."
EPA heartily agrees with another peer reviewer's comments stating that it is difficult to
understand "why we have such an unusual database .... for this chemical." And that "it is
unusual in that there is considerable mechanistic data, but has a less than acceptable toxicology
package."
General line item comments
The specific line by line item comments sent by the peer reviewers were considered and
they resulted in changes where EPA thought appropriate. EPA agreed with most of these
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Parker, August, 1998
Re: DCA Comments
comments. For example, statistical significance values were added to the tabulation of animal
studies, a statement regarding lARC's rinding of animal evidence was added as suggested,
adequacy of the studies due to numbers of animals and the implications this has on study power
was added, as suggested. Other changes were not made because EPA did not believe they were
necessary. In some cases the information could be accessed elsewhere and including it in the
paper would not change the discussion or data interpretation.
Regarding abstracts
It is true, as mentioned by one of the peer reviewers, that some of the references cited are
"only abstracts". This is true, however, these presentations contain important findings and
information highly pertinent to the subject matter of the paper. The EPA author discussed most
of these findings with the investigators and/or attended meeting presentations. Most of the
findings are being published in scientific journal articles. A few authors sent copies of journal
articles in draft or that had been submitted for publication. Some of the articles were already
accepted for publication. These references were added to the final draft paper, in several cases
replacing abstract citations. A few other abstract citations have also been added. EPA
determined that including known findings, although they were published only in abstract form,
was more important than omitting the relevant information, particularly when it was soon to be
published.
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