Professional Documents
Culture Documents
University of North Carolina, Chapel Hill, North Carolina; \Jellinek, Schwartz & Connolly, Inc., Arlington, Virginia;
**American Health Foundation, Valhalla, New York; and van Gemert & Hauswirth,
L.L.C., Charlotte Hall, Maryland 20622
Received October 20, 1998
The current United States Environmental Protection Agency (EPA) classification of di(2-ethylhexyl)phthalate (DEHP) as a B2 probable human carcinogen is based on outdated information. New toxicology
data and a considerable amount of new mechanistic
evidence were used to reconsider the cancer classification of DEHP under EPAs proposed new cancer risk
assessment guidelines. The total weight-of-evidence
clearly indicates that DEHP is not genotoxic. In vivo
administration of DEHP to rats and mice results in
peroxisome proliferation in the liver, and there is
strong evidence and scientific consensus that, in rodents, peroxisome proliferation is directly associated
with the onset of liver cancer. Peroxisome proliferation is a transcription-mediated process that involves
activation by the peroxisome proliferator of a nuclear
receptor in rodent liver called the peroxisome proliferator-activated receptor (PPARa). The critical role of
PPARa in peroxisomal proliferation and carcinogenicity in mice is clearly established by the lack of either
response in mice genetically modified to remove the
PPARa. Several mechanisms have been proposed to
explain how, in rodents, peroxisome proliferation can
lead to the formation of hepatocellular tumors. The
general consensus of scientific opinion is that PPARainduced mitogenesis and cell proliferation are probably the major mechanisms responsible for peroxisome
proliferator-induced hepatocarcinogenesis in rodents.
Oxidative stress appears to play a significant role in
this increased cell proliferation. It triggers the release
of TNFa by Kupffer cells, which in turn acts as a potent mitogen in hepatocytes. Rats and mice are
uniquely responsive to the morphological, biochemical, and chronic carcinogenic effects of peroxisome
proliferators, while guinea pigs, dogs, nonhuman primates, and humans are essentially nonresponsive or
refractory; Syrian hamsters exhibit intermediate re1
INTRODUCTION
327
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Copyright 1999 by Academic Press
All rights of reproduction in any form reserved.
328
DOULL ET AL.
329
TABLE 1
Comparison of DEHP with Other Peroxisome Proliferators: Relative Strength of POX a and CAT b Induction
a
b
Enzyme induction
Peroxisome proliferators
Very weak
Weak
Intermediate
Strong
Very strong
Acetylsalicyclic acid
DEHP
Clofibrate
Nafenopin
WY-14,643
Ciprofibrate
Palmitoyl-CoA oxidation.
Carnitine acetyltransferase.
TABLE 2
Comparison of DEHP with Other Peroxisome
Proliferators: Induction of Relative Liver Weight
Compound
Dose
(mg/kg body wt/day)
DEHA
DEHP
Clofibrate
Nafenopin
WY-14,643
Ciprofibrate
1250
600
250
50
50
12.5
1.36
1.52
1.79
1.97
1.94
1.95
Note. Values from a single study by Takagi et al. (1992) with doses
readjusted to mg/kg body wt/day (Huber et al., 1996).
majority of rodent peroxisome proliferators are nongenotoxic agents, a number of these chemicals have
been shown to increase the incidence of liver tumors in
the rat and/or mouse (Reddy and Lalwani, 1983; Bentley et al., 1993; Ashby et al., 1994).
Because the hepatic morphological and biochemical
effects of DEHP in rats and mice are essentially identical to those produced by a wide range of other rodent
peroxisome proliferators, it is appropriate to utilize all
information on rodent peroxisome proliferators per se
for assessing the risk of DEHP to humans. DEHPinduced hepatic peroxisome proliferation is not due to
the parent diester but to DEHP metabolites. The primary metabolites of DEHP are MEHP and 2-ethylhexanol (2-EH). With MEHP, additional studies have
demonstrated that the proximate peroxisome proliferators are the (v-1)-hydroxy and keto metabolites,
whereas with 2-EH the proximate peroxisome proliferator is EHA (Elcombe and Mitchell, 1986; Lake and
Lewis, 1993; Lhuguenot and Cornu, 1993; Mitchell et
al., 1985b).
Rodent peroxisome proliferators exhibit marked potency differences (Bentley et al., 1993; Lake and Lewis,
1993; Ashby et al., 1994; Lake, 1995a; Huber et al.,
1996). For example, the hypolipidemic agent ciprofibrate is several orders of magnitude more potent than
DEHP, which is somewhat more potent than di-(2ethylhexyl)adipate (DEHA) (Reddy et al., 1986). Calculations from literature data suggest that DEHP and
DEHA are 15 and 5 times more potent, respectively,
than acetylsalicylic acid (Barber et al., 1987). Thus,
based on potency to induce enzyme activities, such as
the peroxisomal fatty acid b-oxidation cycle (e.g., palmitoyl-CoA oxidation) and CAT, DEHP may be considered a relatively weak proliferator (Table 1). The more
potent peroxisome proliferators, such as clofibrate and
nafenopin, have been reported to exhibit lower steeper
doseresponse curves than DEHP (Huber et al., 1996).
In addition, the more potent agents also produce greater
increases in relative liver weight at lower dose levels
(Table 2).
All peroxisome proliferators produce an initial in-
330
DOULL ET AL.
TABLE 3
Comparison of the Hepatocarcinogenic Potential of DEHP
with That of Other Peroxisome Proliferators in Rats
Chemical
% in diet
mg/kg
body wt /day
Duration of study
(months)
% Animals
with tumors
24
010
Control
DEHA
DEHP LOEL
DEHP medium dose
DEHP highest dose
Clofibrate
2.5
0.6
1.2
2.0
0.5
1250
300
600
1000
250
24
24
24
24
1630
4
12
24
6079
4091
Nafenopin
Nafenopin
WY-14,643
Ciprofibrate
0.1
0.2
0.1
0.02
50
100
50
10
1825
13
1416
14
73
65
100
100
Reference(s)
Goodman et al. (1979), NTP (1982a),
Rao et al. (1990)
NTP (1982b)
NTP (1982a)
NTP (1982a)
Rao et al. (1987, 1990)
Svoboda and Azarnoff (1979), Reddy and
Qureshi (1979)
Reddy and Reddy (1977)
Kraupp-Grasl et al. (1990)
Reddy et al. (1979), Rao et al. (1984b)
Rao et al. (1984a)
331
332
DOULL ET AL.
333
334
DOULL ET AL.
TABLE 4
Table of Abbreviations
Abbreviation
Written
PPARa
MOE
NOEL
LOEL
DEHP
EPA
POX
CAT
MEHP
2-EH
DEHA
8-OH-dG
MPG
RXR
mPPARa
IARC
WHO
TGF
PPRE
UDS
SSDB
PEL
STEL
ACGIH
MAK
LADD
335
Evaluation
The effects of peroxisome proliferators on hepatic
peroxisomes and on hepatocyte growth are intimately
linked, and it is not possible to distinguish the relative
contribution of these two responses in the mode of
carcinogenic activity. However, it is generally accepted
that these two effects, when taken together, can completely account for the mode of carcinogenic activity of
peroxisome proliferators in rodent liver (Lake, 1995a,b;
Cattley et al., 1998). These adaptive effects, and ultimately carcinogenicity, are dependent on activation of
gene expression via a nuclear receptor, PPARa. Therefore, it becomes necessary to consider species differences in cellular responses and PPARa to determine if
the mode of action would be reasonably anticipated to
operate in humans.
SPECIES DIFFERENCES IN HEPATIC PEROXISOME
PROLIFERATION
336
DOULL ET AL.
TABLE 5
Examples of Species Differences in Hepatic Peroxisome Proliferation
Species examined
Compound
Responsive
Intermediate
Nonresponsive
Bezafibrate
Rat, mouse
Ciprofibrate
Rat, mouse
Globuzarit
Clofibrate
Di(2-ethylhexyl)phthalate
Rat, mouse
Rat, mouse
Rat, mouse
Syrian hamster,
guinea pig
Syrian hamster,
rabbit
Syrian hamster
Syrian hamster
Syrian hamster
Dehydroepiandrosterone
LY 171883
Rat, mouse
Rat, mouse
Syrian hamster
Syrian hamster
Nafenopin
Oxadiazon
Rat
Rat, mouse
Syrian hamster
Guinea pig
Guinea pig, dog, Rhesus
monkey
Guinea pig, marmoset
Dog
Dog, marmoset
Marmoset, Rhesus monkey
Guinea pig, Cynomolgus
monkey, marmoset
Reference(s)
Watanabe et al. (1989)
Makowska et al. (1992), Graham
et al. (1994)
Orton et al. (1984)
Holloway et al. (1982)
Osumi and Hashimoto (1978),
Lake et al. (1984), Rhodes et al.
(1986), Short et al. (1987)
Sakuma et al. (1992)
Eacho et al. (1986)
Lake et al. (1989)
Richert et al. (1996)
Note. Peroxisome proliferation was assessed by ultrastructural examination and/or measurement of marker enzyme activities. Intermediate species are less responsive than the rat and mouse, whereas nonresponsive species either are refractory or exhibit only a small response
at high dosage. For further details see individual references.
studies include chronic investigations where peroxisome proliferators have been administered for extended
periods without any evidence of either significant peroxisome proliferation or tumor formation (see below).
The majority of studies performed in primates suggests that rodent peroxisome proliferators have little
or no effect in either Old World (e.g., Cynomolgus and
Rhesus monkey) or New World (e.g., marmoset) monkeys (Tucker and Orton, 1988, 1993, 1995; Bentley et
al., 1993; Lake, 1995a,b). However, albeit at high doses,
positive responses with ciprofibrate and DL-040 have
been reported in the Cynomolgus and/or Rhesus monkey (Reddy et al., 1984; Lalwani et al., 1985). The lack
of effect of rodent peroxisome proliferators on primate
liver in in vivo studies is supported by the results of in
vitro studies with cultured primate hepatocytes.
Some primate studies have been conducted with DEHP
and DEHP metabolites. No increase in peroxisome
numbers or associated enzyme activities were observed
in marmosets exposed by oral gavage to DEHP at up to
2000 mg/kg/day for 14 days (Rhodes et al., 1986). More
recently, marmosets exposed for 13 weeks to DEHP at
doses of up to 2500 mg/kg/day showed no treatmentrelated effects on hepatic peroxisomes or peroxisomal
enzymes (Kurata et al., 1998). Similarly, DEHP at
doses of 100 and 500 mg/kg/day for 21 days did not
produce hepatic peroxisome proliferation in the Cynomolgus monkey (Short et al., 1987). In agreement with
the lack of effect of DEHP on primate liver after in vivo
treatment, a number of DEHP metabolites have been
shown not to produce peroxisome proliferation in hepatocyte cultures from marmosets and Cynomolgus
monkeys.
In assessing species differences in hepatic peroxi-
some proliferation, a number of factors must be considered. These include the metabolism, disposition,
and dose of the test compound, sex differences, and
intrahepatic differences in response. Toxicokinetic
studies using clobuzarit, di-n-butylphthalate, 2,4-dichlorophenoxyacetic acid, gemfibrozil, and WY-14,643
have demonstrated that species differences in peroxisome proliferation and/or cell replication do not result
solely from differences in blood concentrations (Cattley
et al., 1998).
Although many studies have demonstrated that the
chronic administration of peroxisome proliferators may
produce liver tumors in the rat and/or mouse, few chronic
studies have been performed in other species. Clobuzarit
has been shown to produce liver tumors in the mouse and
both nafenopin and WY-14,643 produced tumors in both
rats and mice (Reddy and Lalwani, 1983; Tucker and
Orton, 1995), whereas in the Syrian hamster clobuzarit
did not produce tumors after 2 years (Tucker and Orton,
1995) and neither nafenopin nor WY-14,643 produced
tumors after 80 weeks of treatment (Lake et al., 1993a).
In the marmoset, clofibrate (which produces liver tumors
in the rat) did not produce any tumors in a 6 1/2-year
study, which is around half the expected life span of this
species (Tucker and Orton, 1993). Other studies in New
or Old World primates of ;6 month duration have been
conducted with several peroxisome proliferators, including ciprofibrate, clobuzarit, clofibrate, and fenofibrate.
While some increases in liver weight were reported, there
was no evidence of significant peroxisome proliferation or
peroxisome proliferator-induced liver lesions (Blane and
Pinaroli, 1980; Graham et al., 1994; Tucker and Orton,
1993, 1995; Williams and Perrone, 1996).
337
TABLE 6
Peroxisomal Effects of Hypolipidemic Drugs in Human Liver
Compound
Number of
patients
Ciprofibrate
Clofibrate
7
16
Fenofibrate
Fenofibrate
Fenofibrate
Gemfibrozil
8
38
12
9
Effect on peroxisomes a
Increase (30%) in volume density of peroxisomes
Increase (50%) in peroxisome numbers, but not in volume density of
peroxisomes
No change (qualitative)
No change (qualitative)
No effect on either volume density or number of peroxisomes
No change (qualitative)
Reference
Bentley et al. (1993)
Hanefeld et al. (1983)
Blane and Pinaroli (1980)
Blumcke et al. (1983)
Gariot et al. (1987)
De la Iglesia et al. (1982)
Human Studies
Studies in humans have been conducted in patients
treated with several hypolipidemic agents (all rodent
peroxisome proliferators), including ciprofibrate, clofibrate, fenofibrate, and gemfibrozil [reviewed in Bentley
et al., 1993; Ashby et al., 1994; International Agency
for Research on Cancer (IARC), 1995, 1996; Williams,
and Perrone, 1996; Cattley et al., 1998]. While most
studies with fenofibrate and gemfibrozil (Blane and
Pinaroli, 1980; De la Iglesia et al., 1982; Blumcke et al.,
1983; Gariot et al., 1987) failed to detect any changes in
peroxisomes in human hepatocytes, some morphological effect was noted in one study with clofibrate and in
one study with ciprofibrate (Table 6). In the clofibrate
study, morphometric analysis revealed a small increase in the number of peroxisomes, but no significant
change in the volume density of peroxisomes (Hanefield et al., 1983). As volume density is a more sensitive
indicator of peroxisome proliferation than peroxisome
number, the significance of this study is questionable
(IARC, 1995; Huber et al., 1996). In a study with ciprofibrate, a small 30% increase in the proportion of
hepatocyte cytoplasm occupied by peroxisomes was observed (reviewed in Bentley et al., 1993). As in other
studies, considerable interindividual variation in peroxisome morphometrics was observed, which together
with cell-to-cell and lobular variations makes it difficult to attach any clear biological significance to these
findings. Thus, humans, like nonhuman primates, are
refractory to peroxisome proliferation. This is in accord
with the low expression in humans of PPARa, which
mediates peroxisome proliferation by peroxisome proliferators.
Several epidemiological studies have examined the
association of cancer with the use of hypolipidemic
drugs which are peroxisome proliferators in rodents.
None has reported a clear association. In 1978, the
World Health Organization (WHO) reported the results of a randomized trial to determine whether clofibrate treatment would lower the incidence of ischemic
heart disease in men (WHO, 1978). This study reported
a nonsignificant excess of deaths from cancer in treated
338
DOULL ET AL.
TABLE 7a
Some Examples of Species Differences in the Effects of Rodent Peroxisome
Proliferators in Primary Hepatocyte Cultures a
Species
Compound
Beclobric acid
Benzbromarone
Bezafibrate
Ciprofibrate
Clofibrate
Clofibric acid
2-Ethylhexanol
2-Ethylhexanoic acid
Fenofibric acid
Fomesafen
LY171883
MEHP
MEHP metabolite VI
MEHP metabolite IX
Methylclofenapate
Mono-(2-ethylhexyl)
adipate and derivatives
Monoisodecyl and
monoisononyl phthalates
Nafenopin
Oxadiazon
Propaquizafop and free
acid derivative
Trichloroacetic acid
Wy-14,643
Rat
Mouse
Guinea pig
Marmoset
Cynomolgus
monkey
Rhesus
monkey
Human
Reference
Yes b
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ND
ND
ND
Yes
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Yes
Yes
ND
ND
ND
Yes
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No
ND
ND
ND
ND
ND
ND
ND
No
ND
ND
No
No
No
No
No
No
ND
ND
ND
No
No
No
No
No
No
No
ND
ND
ND
No
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No
No
ND
ND
ND
No
ND
ND
ND
ND
No
No
ND
ND
ND
ND
No
No
ND
ND
ND
No
ND
No
ND
No
ND
ND
No
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No
ND
No
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No
ND
ND
ND
ND
ND
ND
ND
ND
ND
No (3) c
ND
No (3)
ND
ND
No (1)
ND
No (1)
No (3)
No (3)
No (1)
No (3)
ND
No (6)
ND
ND
No (3)
ND
No (3)
No (3)
ND
No (3)
No (2)
ND
No (3)
No (3)
No (3)
ND
No
No (3)
Yes
Yes
No
No
ND
ND
ND
Yes
Yes
Yes
ND
ND
ND
ND
ND
ND
No
No
ND
ND
ND
ND
ND
ND
ND
ND
ND
No (6)
Yes
Yes
Yes
Yes
Yes
Yes
ND
ND
No
ND
No
ND
No
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No (2)
No (3)
No (2)
a
Effects of test compounds were assessed by changes in enzyme activities (e.g., palmitoyl-CoA oxidation, lauric acid 12-hydroxylase) and/or
organelle proliferation.
b
Species response defined as yes, significant concentration-related response; no, no effect/little effect; ND, not determined.
c
When given in the reference, the figures in parentheses represent the numbers of different human hepatocyte preparations examined.
339
TABLE 7b
Some Examples of Species Differences in the Effects of Rodent Peroxisome
Proliferators in Primary Hepatocyte Cultures a
Species
Compound
Rat
Mouse
Syrian
hamster
Guinea
pig
Marmoset
Human
Reference
Ciprofibrate
2-Ethylhexanoic acid
Fomesafen
Methylclofenapate
Yes b
Yes
Yes
Yes
Yes
Yes
ND
Yes
ND
ND
ND
ND
ND
ND
ND
ND
Yes
ND
ND
ND
ND
ND
ND
ND
ND
No
ND
ND
ND
ND
No
ND
ND
ND
No
ND
ND
ND
ND
ND
ND
ND
Yes/no d
ND
ND
No (4) c
No (3)
No (3)
No
No (3)
ND
ND
ND
No (5)
Nafenopin
Effects of test compounds were assessed by measurement of the hepatocyte labeling index or incorporation of [ 3H]thymidine into DNA.
Species response defined as yes, significant response; no, no effect/little effect, ND, not determined.
c
When given in the reference, the figures in parentheses represent the numbers of different human hepatocyte preparations examined.
d
Replicative DNA synthesis was increased in the absence but not in the presence of serum. However, no effect on peroxisome proliferation
was noted.
b
produce any significant peroxisome proliferation in human hepatocytes. This is consistent with the small
number of PPARa found in human livers. A large number of human subjects have been treated for relatively
long periods of time with hypolipidemic drugs which
are known potent rodent peroxisome proliferators. The
potential carcinogenic risk of hypolipidemic therapy
with clofibrate and gemfibrozil has been evaluated in
two, albeit limited, clinical trials (reviewed in Bentley
et al., 1993; IARC, 1995, 1996). Both these studies and
other human studies with lipid-lowering drugs showed
no indication of any increase in cancer associated with
long-term exposure to hypolipidemic peroxisome proliferators. Indeed, when recently evaluated by IARC
(1996), both clofibrate and gemfibrozil were placed in
group 3 (i.e., the agent is not classifiable as to its
carcinogenicity to humans).
In Vitro Studies in Cultured Hepatocytes
Studies in various laboratories have demonstrated
that primary hepatocyte cultures represent a validated
in vitro model for studying various aspects of hepatic
peroxisome proliferation including compound potency
comparisons and evaluation of species differences in
response (Lake and Lewis, 1993; Lake et al., 1993b;
Bentley et al., 1993; Bieri, 1993; Foxworthy and Eacho,
1994; Ashby et al., 1994). Many of the known characteristics of peroxisome proliferation in vivo, including
increased number and size of peroxisomes, differential
induction of peroxisomal enzyme activities, and stimulation of replicative DNA synthesis, have been observed in cultured rat and mouse hepatocytes treated
with peroxisome proliferators (Gray et al., 1983; Bieri
et al., 1984; Elcombe, 1985; Elcombe and Mitchell, 1986;
340
DOULL ET AL.
TABLE 8
The Distribution of PPARa in Rat and Human Tissues within Species, Not across Species e
Species
Method a
Liver
Brain
Lung
Heart
Muscle
Kidney
Pancreas
Adipose
Reference b
Rat
Rat
Human
Human
Human
A
B
C
C
D
Low/high
High
High
Low
18 f
None
Low
Low
None
ND
ND d
Low
Low
Low
ND
None/mod c
High
High
None
ND
ND
High
High
High
5
High
High
High
Mod
5
Low
ND
High
None
ND
None/mod
ND
ND
ND
1
A
B
B
C
D
Note. In this table comparisons of relative PPAR expression levels are only valid for tissues within a species and not a cross species.
a
A, in situ hybridization/immunocytochemistry; B, RNAase protection; C, Northern blotting; D, RT-PCR.
b
A, Braissant et al. (1996); B, Mukhejee et al. (1994); C, Tugwood et al. (1996); D, Auboeuf et al. (1997).
c
Moderate.
d
Not determined.
e
Data also reported in Lemberger et al. (1996).
f
Attomole PPAR RNA/mg total RNA.
in Syrian hamster hepatocytes and neither methylclofenapate nor nafenopin stimulates replicative DNA
synthesis in guinea pig hepatocytes (Elcombe and Styles,
1989; James and Roberts, 1996). The effect of rodent
peroxisome proliferators on replicative DNA synthesis
in primate hepatocytes does not appear to have been
extensively investigated. In one study nafenopin was
reported to increase replicative DNA synthesis in marmoset hepatocytes when cultured in the absence, but
not in the presence, of serum (Bieri et al., 1988). The
significance of this finding is uncertain as these workers reported that nafenopin did not produce peroxisome proliferation (as assessed by a lack of effect on
palmitoyl-CoA oxidation activity and organelle numbers) in cultured marmoset hepatocytes.
A number of investigations have demonstrated that
peroxisome proliferators may inhibit the rates of basal
and/or transforming growth factor-b 1 (TGF-b 1)-induced
apoptosis in cultured rodent hepatocytes (James and
Roberts, 1996; Roberts, 1996; Perrone et al., 1998). By
using the terminal transferase dUTP biotin nick endlabeling technique, Perrone et al. (1998) demonstrated
that ciprofibrate and clofibrate had no effect on TGFb 1-induced apoptosis in human hepatocytes. In contrast, both peroxisome proliferators significantly increased the basal rate of apoptosis in cultured human
hepatocytes. These authors concluded that there were
differences between rat and human hepatocytes in the
effects of peroxisome proliferators on DNA synthesis
(see above) and apoptosis. As such, their data support
the hypothesis that human liver cells are refractory to
peroxisome proliferator-induced hepatocarcinogenesis
(Perrone et al., 1998). Unlike rodent hepatocytes, where
apoptosis may be suppressed, human hepatocytes respond to peroxisome proliferators with an increase in
apoptosis, which should be anticarcinogenic.
In conclusion, studies of in vitro responses to peroxisome proliferators have been consistent with reported
species differences in PPARa expression and in vivo
responses to these agents. The induction of key biomarkers of peroxisome proliferation, including fatty acidoxidizing enzyme activities (e.g., palmitoyl-CoA oxidation, acyl-CoA oxidase, lauric acid 12-hydroxylase),
organelle proliferation, and replicative DNA synthesis
has been clearly demonstrated in rodent hepatocytes
following in vitro treatment with a range of peroxisome
proliferators. The overall induction of fatty acid-oxidizing enzyme activities and/or organelle proliferation in
human hepatocytes has been reported to be weak in
one study and essentially nonexistent in other studies
with a range of 13 rodent peroxisome proliferators.
Moreover, the induction of replicative DNA synthesis
in human hepatocytes has been consistently negative
in studies with five rodent peroxisome proliferators.
Such agents also appear to exhibit different effects on
apoptosis in rodent and human hepatocytes. These findings clearly strengthen the conclusion that humans are
341
342
DOULL ET AL.
Evaluation
There is clear evidence for species differences in xenobiotic-induced hepatic peroxisome proliferation and
cell replication. Peroxisome proliferators produce marked
biochemical and morphological changes in rat and mouse
hepatocytes and following prolonged exposures may
also produce liver tumors. Species other than the rat
and mouse are clearly less responsive or refractory to
peroxisome proliferators. The available data suggest
343
TABLE 9
Summary Listing of Genotoxicity Data
for DEHP and MEHP
Assay
Prokaryotic mutation assays
Salmonella
Other bacteria
Yeast/fungus genotoxicity assays
Gene mutation
Mitotic recombination
Aneuploidy
Drosophila mutation assays
Mammalian DNA damage assays
Binding of radiolabeled chemical
32
P-postlabeling assay
Single-strand DNA breaks
Unscheduled DNA synthesis
Mammalian mutation assays
Chinese hamster ovary cells
L5178Y mouse lymphoma cells
Syrian hamster embryo cells
Hamster V79 cells
Human lymphoblast cells
Mammalian chromosome damage assays
Rodent in vitro chromosomal damge
Aberrations
Aneuploidy
Human in vitro chromosomal damage
Rodent in vivo chromosomal aberrations
Rodent sister chromatid exchange
Human sister chromatid exchange
In vitro rodent micronucleus
In vivo rodent micronucleus
In vivo co-recombinogenesis
Mammalian cell transformation
C3H1OT1/2 cell
SHE cell
BALB/c-3T3 cells
Mammalian germ cell mutation assays
Dominant lethal
DEHP
MEHP
2
2
1/2, 2
1/2, 2
1/2, 2
2
1?
1/2, 2
ND
2
2
2
2
2
ND
ND
2
2
2
?
2
2
ND
2
?
?
ND
2?
1
2
2?
2?
2
2
2
1
1
ND
ND
2?
1?
ND
ND
ND
1/2
1
2
ND
ND
ND
1?
ND
humans exposed daily to therapeutic doses of more potent rodent peroxisome proliferators for extended periods
of time (IARC, 1995, 1996).
GENOTOXICITY
344
DOULL ET AL.
Alternative Approach
EPAs Proposed Guidelines for Carcinogen Risk Assessment (EPA, 1996) allow consideration of several
alternative methods for analyzing carcinogen dose
response data and for extrapolating the effects observed at relatively high doses in laboratory animals to
predict those that might occur at much lower doses
relevant to human exposure. Thus, in contrast to the
Based on the established association of DEHP-induced rodent liver cancer with a receptor-mediated
mechanism and general scientific consensus that receptor mechanisms involve dose thresholds (Poland,
1997), it is concluded that risk assessment with DEHP
can most appropriately be conducted using the MOE
approach.
345
TABLE 10
Morphological, Biochemical, and Tumorigenic Observations from DEHP Bioassays
with Rats and Mice (David et al., 1996, 1997)
F344 Rats
Group/dose
100 ppm
7.3 mg/kg/day (F)
5.8 mg/kg/day (M)
500 ppm
36.1 mg/kg/day (F)
28.9 mg/kg/day (M)
2500 ppm
181.7 mg/kg/day (F)
146.6 mg/kg/day (M)
12500 ppm
938.5 mg/kg/day (F)
789.0 mg/kg/day (M)
B6C3F1 Mice
Observation
No
No
No
No
Group/dose
treatment-related effects
organ weight changes
histopathological changes
detectable peroxisome prolifer
100 ppm
No treatment-related effects
No organ weight changes
No histopathological changes
No detectable peroxisome prolifer
NOEL: Peroxisome proliferation
Increased peroxisome proliferation
Increased liver weight
Increased incidence of hepatocellular
adenomas (males only)
NOEL: Carcinogenic potential
Decreased survival
Increased peroxisome proliferation
Increased liver weight
Increased incidence of hepatocellular
adenomas and carcinomas
500 ppm
Observation
No treatment-related effects
No organ weight changes
No histopathological changes
No detectable peroxisome prolifer
NOEL: Peroxisome proliferation
Increased peroxisome proliferation
Increased liver weight (males only)
No histopathological changes
NOEL: Carcinogenic potential
Increased peroxisome proliferation
Increased liver weight
Increased incidence of hepatocellular
adenomas and carcinomas
Decreased survival
Increased peroxisome proliferation
Increased liver weight
Increased incidence of hepatocellular
adenomas and carcinomas
346
DOULL ET AL.
GENERAL POPULATION
The major source of exposure for the general population is via food residues arising from migration of
DEHP into food from plastic containers. In general,
food residues do not exceed 1 mg/kg (1000 mg/kg) of
food, although considerably higher residues may be
associated with fatty foods such as oils, milk, cheese,
meat, and fish. The magnitude of the residues will
depend on the DEHP content of the container/wrapper
material and the duration and conditions (temperature) of storage. The U.S. FDA has established a limit
of 3% DEHP content for all materials used to store
foodstuffs. It is generally estimated that the total daily
intake of DEHP in food products ranges from about
0.27 mg/day (3.8 mg/kg/day) to a high of about 2.0
mg/day (30 mg/kg/day) (Huber et al., 1996).
In comparison with possible intakes from food, DEHP
exposures from ambient air and drinking water are very
small (Huber et al., 1996) and make an insignificant
contribution to the total daily intake of the general population.
OCCUPATIONAL EXPOSURES
347
TABLE 11
Use Information for Medical Devices Containing DEHP
Use information
Product
category
Selected use
scenario
No.
units/day
Replacement
time
Treatment regimen
Comments
iv blood sets
Transfusion of
platelets in
leukemia
patients
1 per session
70 times/year for 2
years (Jacobson et
al., 1977)
Hemodialysis
sets
Short-term and
long-term
hemodialysis
Typically 5 h (Flaminio
et al., 1988; Lewis et
al., 1978)
1 per session
Autopheresis
sets
Donation of plasma
and return of
RBCs
60 min (assumption)
1 per session
Nasogastric
tubes
Enteral feeding
tubes
2 days
(assumed)
24 h
The clear outlier of 649.5 mg for one bag type was discarded from
the original data set.
Thus, for leukemia patients, the treatment period exposure (E T) and LADD are
13 mg DEHP
3 ~70 treatments/year! 3 ~1000 mg/mg!
ET 5
~365 days/year!
5 2,500 m g/person/day
LADD 5
348
DOULL ET AL.
Most of the DEHP exposure values from the Pollack et al. (1985) study
clustered in the 23.8 to 98.9 mg range; if the two extreme high values in
the data set (289 and 360 mg) are excluded as statistical outliers, a
mean of 56.6 mg is obtained, with a standard deviation of 33 mg.
74 mg DEHP/treatment
3 ~24 treatments/year! 3 ~1000 mg/mg!
ET 5
~365 days/year!
5 4900 mg/person/day
LADD 5 ~4900 mg/person/day!
3 ~0.167 years/70 years!
5 12 mg/person/day
and for long-term dialysis patients
74 mg DEHP/treatment
3 ~156 treatments/year! 3 ~1000 mg/mg!
ET 5
~365 days/year!
5 32,000 mg/person/day
LADD 5 ~32,000 mg/person/day! 3 ~15 years/70 years!
5 6900 mg/person/day.
AUTOPHERESIS SETS
349
5 487 mg/person/day
LADD 5 ~487 mg/person/day! 3 ~5 years!/~70 years!
5 35 mg/person/day.
NASOGASTRIC FEEDING TUBES
5 4200 mg/day.
The LADD for a trauma patient using the device for 12
days is
LADD 5
350
DOULL ET AL.
DEHP are much lower than those of workers, the major source of exposure, food, leading to oral intakes in
the range of 330 mg/kg body wt/day. Ingestion of water
and inhalation of ambient air constitute negligible
sources of exposure to DEHP.
In contrast, nonoccupational exposures from medical
devices are significant and in some cases may approach
those of workers. Of several devices evaluated, maximum exposures are encountered by long-term hemodialysis patients. These individuals receive up to about
457 mg/kg body wt/day of DEHP over the course of a
typical 1-year treatment period and this equates to a
LADD of about 99 mg/kg body wt/day when estimated
on the basis of a possible 15-year dialysis period in a
70-year life span. Hemodialysis patients constitute a
relatively small patient subpopulation and more common exposures from medical devices result, for example, from the use of transfusion equipment (iv sets and
blood bags). Even with fairly frequent use of transfusions, however (e.g., 70 treatments/year for leukemia
patients), the exposures are much lower and do not
exceed about 36 mg/kg body wt/day (LADD of about 1
mg/kg body wt/day for a 2-year transfusion period in a
70 year life span).
In considering the potential health relevance of human exposures to DEHP it is important to take into
account the route as well as the level of exposure. As
discussed in the pharmacokinetics section of this article, the enzymatic breakdown of DEHP to MEHP is of
critical importance because, in rodents, MEHP and
several of its metabolites are the active peroxisome
proliferators; DEHP is not, itself, an active peroxisome
proliferator. Since the conversion of DEHP to MEHP
occurs almost exclusively in the gastrointestinal tract
(the lipase responsible is secreted into the small intestine from the pancreas), the activation of DEHP is
limited mainly to exposures via the oral route. Only
low titers of DEHP-hydrolyzing enzymes are present in
the liver and other tissues (Albro, 1986; Huber et al.,
1996) so that DEHP entering the systemic circulation
by the inhalation, dermal, or intravenous routes avoids
contact with the lipase present in the gut and is not
converted to an active peroxisome proliferator. Clearly,
this has important toxicokinetic consequences because,
even if humans were sensitive to MEHP (which they
are not), circulating levels of MEHP and other active
metabolites would be extremely low in humans undergoing transfusion or dialysis procedures (Rock et al.,
1978; Chu et al., 1978). The only possible exception to
this is if, as is apparently the case under certain conditions, DEHP is converted to MEHP to any significant
extent during storage of blood or blood products.
MOE Approach
MOE values for the various exposure scenarios discussed and based on the mouse NOEL for peroxisome
TABLE 12
MOEs for Selected Routes of Human Exposure
Exposure population/route
General population/oral
Daily ingestion in food
Occupational/inhalation
OSHA, ACGIH (PEL, TLV)
LADD (40-year work-life)
Medical devices/iv and other
Transfusion
Intensive (12 years)
LADD (2 years/lifetime)
Hemodialysis
Over 1 year
LADD (15 years/lifetime)
Level of exposure a
(mg/kg body wt/day)
MOE b
Up to 30
667
700
280
29
71
36
1
556
20,000
457
99
44
202
The exposure values given represent worst-case scenarios. Actual exposures are likely to be very much lower than these values.
b
The MOE (margin of exposure) values are obtained with reference to the mouse NOEL of 20 mg/kg/day for peroxisome proliferation.
351
are more sensitive to the adverse effects of the chemical than the test animals. This assumption is clearly
quite contrary to all available data on the action of
DEHP as a liver carcinogen. Consequently, it is concluded that the acceptable MOE for DEHP can justifiably be reduced by a factor of 10 from the usual default
value.
Human variability in sensitivity to the phenomenon
involved. There is no evidence to suggest any significant variation in sensitivity to DEHP or other peroxisome proliferators within the human population (ATSDR,
1997). As indicated above, humans are extremely nonresponsive to the biochemical, morphological, and
other effects of DEHP and other peroxisome proliferators. It seems likely that, if they exist, some evidence of
genetic polymorphism or ethnic variation would have
been uncovered in the clinical and epidemiological
studies that have been conducted with hypolipidemic
drugs; to date, this has not been the case. While this
does not provide unequivocal proof that such polymorphic variation does not exist, it suggests a low probability of occurrence and an insignificant difference in
phenotype expression. It must be concluded that currently available data are inadequate to justify moving
away from the default factor of 10 associated with
intraspecies variation.
Persistence of DEHP in the body. DEHP is rapidly
metabolized and excreted from the body (plasma halflife for DEHP is 10 18 h, while that for MEHP is 3 6
h) and there is no evidence of persistence or tissue
accumulation following repeated daily exposures.
Other factors. Because of the critical importance of
the route of exposure in the metabolic activation of
DEHP, it is concluded that, even though relatively
high, human occupational inhalation exposures and
medical intravenous exposures have little or no relevance to any potential effects that might be mediated
through peroxisome proliferation. Exposure by any
route other than ingestion will not be expected to have
an effect because it bypasses the gastrointestinal conversion of DEHP to MEHP that is required to generate
an active peroxisome proliferator.
This clearly indicates that the only relevant DEHP
exposures likely to affect the general human population are residues in food. As discussed, these are small,
about 30 mg/kg body wt/day, and are associated with a
MOE of almost 700.
Based on the total weight-of-evidence available, it is
concluded that 10 constitutes an acceptable and highly
conservative MOE for purposes of assessing the potential human risks associated with exposure to DEHP.
This conclusion has been reached by procedures that
are consonant with those outlined in EPAs Proposed
Guidelines for Carcinogen Risk Assessment.
352
DOULL ET AL.
CONCLUSIONS
is almost 700. This exceeds by a factor of 70 an acceptable MOE obtained by a MOE analysis conducted according to the Proposed Guidelines for Carcinogen Risk
Assessment.
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