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Part 4 - Science Policy of the document titled ALPHA-2u-GLOBULIN:
ASSOCIATION WITH CHEMICALLY INDUCED RENAL TOXICITY AND NEOPLASIA
IN THE MALE RAT has recently been completed. A copy is attached for your
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PART 4. SCIENCE POLICY
I. Background and Introduction
An increased incidence of neoplasms in chemically tested
animals is customarily viewed by scientists as an indication of
carcinogenicity in animals and as some signal that humans may be
similarly affected. From this line of reasoning, EPA generally
presumes that animal tumor findings indicate there may be a cancer
hazard to humans, although a final judgment as to human
carcinogenic potential can be made only in relation to all other
relevant information. There has been growing interest in recent
years in the possibility that tumors produced in the tubule of the
male rat kidney subsequent to the accumulation of the low-
molecular-weight protein alpha-2u-globulin (a2(j-g) , might involve
a process that occurs only in the male rat. Because of the
implications to cancer risk assessment, the Risk Assessment Forum
established a Technical Panel to examine the available information
on CL, -g accumulation in the kidney, associated renal disease, and
kidney cancer. These findings are reported in parts 1-3 of this
report.
This part 4 provides guidance to EPA risk assessors regarding
evaluation of renal tubule tumors in the male rat and presents
Forum conclusions regarding potential human hazard and risk for a
special subset of renal cancer, those renal tubule tumors appearing
to arise as a result of chemically induced a2u-g accumulation in the
male rat kidney.
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II. Basis for Science Policy on Male Rat Kidney Tumors
The scientific data supporting the Technical Panel's
conclusions regarding the a2u-g sequence of lesions1 are covered in
depth in the preceding sections (parts 1-3) of this document. The
information below highlights critical data and outlines inferential
bridges used to select the most plausible explanation for the
information available on male rat kidney tumors.
A. Low-molecular-weight proteins in the rat
In the rat kidney, as in those of other mammals, low-
molecular-weight proteins are removed from the plasira to the urine
by glomerular filtration followed by partial reabsorption from the
urine into the renal tubule of the kidney with subsequent
catabolism (destructive metabolism). One of these low-molecular-
weight proteins, Q2u-g produced by the liver under the stimulus of
testosterone, reaches very high levels in the plasma and urine of
young adult male rats, gradually declining with age.
Alpha-2u-globulin is regarded as a member of a large
superfamily of proteins thought to be carriers of lipophilic
(affinity for fat) molecules. Some of these proteins, e.g.
retinol-binding protein and lactoglobulins, are found in man;
species, including humans. Others, like a2u-g, are found only in
selective species. The only member of the protein superfamily with
a clearly defined physiological role is retinol-binding protein,
the carrier protein for vitamin A. Although these low-molecular-
1 Alteration is structural or functional, due to disease;
commonly limited to morphological alterations.
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weight proteins are believed to have a similar three-dimensional
structure, the alignment of amino acid residues (sequence homology)
between any pair of proteins in the superfamily is small, roughly
20 percent. The exception is a2u~9 and mouse major urinary
protein(s) (MUP) which share approximately 90 percent sequence
homology.
Alpha-2u-globulin derived from hepatic synthesis is not known
to occur in the female rat or any other species, including humans.
Although similar forms of o:2 -g are synthesized at nonhepatic sites
in female rats and in the male NCI Black Reiter rat (NBR), a strain
whose males lack hepatic synthesis of a2u~9/ none of these other
forms of a2ij-g nor MUP accumulate in the renal tubule following
administration of the compounds discussed in the Technical Panel
report.
B. Description of the progression from chemically induced alpha-
2u-globulin accumulation to nephropathy and neoplasia
1. Overview
The information available provides a plausible, although
probably incomplete picture of a sequence of events occurring in
the male ra ~ kidney following chemical administration. This
sequence can „•.-.i portrayed on a molecular and cellular level.
Initially, the test chemical appears to bind reversibly to a2u-g,
seemingly forming a complex more resistant to lysosomal
degradation2 than the unreacted protein itself. This shifts the
2Lysosomes are intracellular bodies present mostly in the
liver and kidney. They contain hydrolytic enzymes responsible for
the catabolism of a2(j-g anc^ other proteins.
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balance between reabsorption and catabolisiu and appears to result
in accumulation of the protein complex in a specific segment (P2)
of the renal tubule. Continued compound administration results in
a cytotoxic response from the sustained protein overload to the
renal tubule, causing single cell necrosis (death) of cells lining
the surface of the tubule and other kidney pathology. Dead cells
are replaced by cell division. As the cycle of cell death and cell
replacement continues, tubule cells increase in number
(hyperplasia), and with time neoplasia may occur. It is presumed,
but certainly not proven, that continued cell proliferation plays
a role in the neoplastic process.
The morphological basis for the sequence of events beginning
with an increase in number and size of hyaline droplets3 containing
a2u-g is the demonstration of a progression of characteristic
lesions. Single cell necrosis in the renal tubule can be confirmed
by observation of granular casts.4 Enhanced cell replication in
response to cell death can be seen as increased cell division or
can be demonstrated by labeling techniques that measure increased
DNA synthesis. In chronic laboratory animal bioassays involving
administration of compounds that induce a2u-g accumulation, tubule
hyperplasia, linear mineralization in the renal papilla (possibly
3Spherical inclusions in the cytoplasm that, may contain
various proteins.
4The granular casts are composed of sloughed cell debris from
the dead cells. They accumulate at the junction between the P3
segment of the proximal tubule and the descending thin loop of
Henle where diameter is constricted.
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representing remnants of debris from disintegrating granular
casts), and renal tubule tumors are observed.
2. Evidence supporting the specificity of the sequence to
the male rat
Hypothesis-testing experiments conducted over the last decade
in various laboratories have shown a remarkable consistency of
results indicating that chemically induced accumulation of a2u-g in
the male rat kidney can lead to renal tubule tumors. This
information establishes an association between exposure of the male
rat to compounds that induce a, -g accumulation and a specific form
of nephropathy (kidney disease), and it supports an association
between this nephropathic response and renal tubule tumors.
The male rat has consistently responded to administration of
compounds that cause the accumulation of abnormal amounts of a2u-g
in the P2 segment of the renal tubules with a characteristic
nephropathy. The severity of the nephropathy is dose-dependent,
not only with respect to the amount of compound administered, but
also with respect to the concentration of a,u-g in the kidney.
Alpha-2u-globulin nephropathy also differs sufficiently from
chronic progressive nephropathy cor-nonly found in aging male rats
so that the two effects can be differentiated.
In contrast to the response of male rats to compound
administration, mice and female rats administered a2 -g-inducers
under the same conditions did not develop lesions characteristic of
a2u~g nephropathy. When exposed to a2u-g-inducers in chronic
bioassays, these latter animals did not develop an increased
incidence of renal tubule tumors, even though male rats developed
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a dose-dependent neoplastic response in the kidney. The
specificity of the male rat response has also been tested to a
limited extent in a number of other species, with no evidence of
protein droplet nephropathy in dogs, guinea pigs, hamsters, or
monkeys. Since these species (and the mouse and female rat) have
proteins similar in structure to a2u-g, their lack of nephrotoxic
(damage to kidney cells) response is consistent with the
presumption that the unique a2u-g produced by the liver of male rats
is the necessary determinant for the expression of the renal
effects.
Hyaline droplets in the proximal tubule of untreated male rats
contain a2(j-g, especially in young adults. Hyaline droplets are
substantially reduced in castrated male rats, further indicting the
dependence of this phenomenon on male hormone levels. In female
rats of any age, an observation of protein droplets is rare and
a2y-g is not involved.
Specialized studies involving hormone manipulation have shown
that the development of the early features of a? -g nephropathy is
dependent on the presence of the hepatic form of a2 -g. (1)
Hyaline droplet or Q2u-g accumulation does not occur when a2u-g-
inducers are administered to immature or old male rats that produce
little a2u~9 ^-n ^e liver- (2) Hyaline droplet accumulation is
observed from administration of a2u~g-inducers even in castrated
rats, but the severity of the effect is diminished. (3) Estrogen
administration to male rats reduces the seveirity of a2U~^
nephropathy. (4) Female rats administered an a2u-g-inducer along
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with G2u-g purified from male rat urine clearly showed hyaline
droplet formation, <22u-g accumulation in the kidney, and some
nephropathy even though control female rats show no measurable
effects.
Male rats of the NBR strain provide a unique opportunity for
testing the a2u-g hypothesis since this animal has no detectable
levels of hepatic messenger RNA for a2u~g. Under conditions of
exposure that produced a2u"~g nephropathy in male rats of other
strains, several chemicals administered to the NBR rat did not
induce detectable accumulation of &2u~g in the renal tubules.
Additional experimentation using a nitrosamine as the
initiator of cancer and an a2u-g-inducer as the promoter also
support the observation that a2ij-g -*-s involved in the process
leading to renal tubule tumors in the male rat. In one initiation-
promotion study, the promotion potential of the a2u~g-inducer was
compared and contrasted in male Fischer and NBR rats. Consistent
with the hypothesis that <-'2(J-g is necessary to induce a response,
the promoter did not enhance renal tubule tumor formation in the
a2u-g-def icient NBR rat, but it did promote renal tubule tumor
formation in the Fischer rat.
It is clear that not all renal tubule cancer in laboratory
animals occurs through the hypothesized a2u-g sequence. Other
inducers of rodent renal tubule cancer are well known. These
include, for example, certain nitrosamines in the rat and mouse and
diethylstilbestrol in hamsters. In general, these prototypic renal
carcinogens are active in both males and females. The acute
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nephrotoxic changes in the renal tubules include mild lipid droplet
accumulation and scattered single cell necrosis., but hyaline
droplet accumulation and its specific associated naphropathy are
not characteristic.
Based on the information available to date; about a2u-g-
inducers, these compounds may have additional features that help to
distinguish them from some other chemicals, such as the
nitrosamines, that are also inducers of rodent kidney tumors.
Alpha-2u-globulin inducers identified to date appear to be non-
genotoxic, or only marginally so, suggesting that the mechanism for
tumor induction may not depend on direct genetic injury. So far,
the incidence of renal tumors produced in the male rat has been
relatively low, occurring late in life, and metastasizing rarely.
Distribution studies of compounds and information on chemical
binding to Q2u-g indicate that, of the total chemical administered
to the animal, only a small portion of the metabolites (possibly
the parent compound) appear responsible for inducing a2u~%
accumulation. Considerable amounts of the chemical and other
metabolites may be present in the male rat kidney in apparently
unbound form. These other moieties can, at times, cause toxic
effects in the kidney, including cancer, that are unrelated to the
accumulation of a2u-g. Such information does not preclude a
determination that the a2u-g sequence is involved in some manner
with the renal tumor response.
III. Science policy statement
Based on interpretation of current data, the Technical Panel
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made the following three conclusions. First, the sequence of
events proposed to link &2u-g accumulation to nephropathy and renal
tubule tumors in the male rat is plausible, although not totally
proven.
Second, the a2u~g response following che 'ical administration
appears to be unique to the male.rat. Even though closely related
proteins are present in other species, there is ruo evidence that
they respond to chemical administration in a manner similar to the
male rat.
Third, the response seen in the male rat kidney following
chemically induced a2u~g accumulation is probably not relevant to
humans.
Given the Technical Panel's findings, EPA science policy
regarding use of male rat renal tubule tumors attributable to
individual chemicals or chemical mixtures for human risk assessment
is as follows.
(1) Male rat renal tubule tumors arising as a result of a
process involving a2u-g accumulation do not contribute to
the qualitative weight-of-evidence that a chemical c ozes
a human carcinogenic hazard. Such tumors are r : ~
included in dose-response extrapolations for the
estimation of human carcinogenic risk.
(2) If a chemical induces a2u-g accumulation in male rats,
the associated nephropathy is not used as an endpoint for
determining non-carcinogenic hazard. Estimates of non-
carcinogenic risk are based on other endpoints.
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On the other hand, a2u-g-related kidney tumors do not negate
the consideration of tumors in other sites in the male rat or
tumors in other species. Chemicals can influence cells of various
organs and tissues in different ways. Thus, the determination of
the relevance of tumors at other sites for hazard identification
and the characterization of risk from these tumors proceeds on
their own merits and is not influenced by judgments about the
applicability of the Q2u-g process to the evaluation of renal tubule
tumors in male rats. Likewise, the analysis of the role of
chemically-induced a2u-g accumulation proceeds on its own merits and
is not influenced by determinations made regarding tumors at other
sites.
To determine the applicability of EPA's science policy,
chemicals inducing renal tubule tumors in the male rat are grouped
into one of the following three categories.
(1) The a2u~g seguence of events apply.
(2) Other potential carcinogenic processes apply.
(3) The Q2u-g-associated events occur in the presence of
other potential carcinogenic processes.
IV. Guidance for evaluating el smically-induced male, rat renal
tubule tumors
To determine which of the three categories described above
apply for a given chemical that has produced renal tubule tumors in
the male rat two questions need to be answered. The first is
whether or not a2u-g-associated events are involved in the tumor
development. The second, given an affirmative answer to the first,
is to determine if other processes may also account for the
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demonstrated tumor increase. Guidance for answering these two
questions follows.
A. Renal tubule tumors in male rats and c?2u-g accumulation
Three kinds of information from adequately conducted studies
are needed on a chemical producing renal tubule tumors in the male
rat to ascertain if the <*2u-g process is operative or not.
Affirmative responses define the minimal amount of data needed to
determine that the a2u-g-related sequence pertains. In their
absence, it would be concluded that the <22u~cj process is not
operative. The three components for making these determinations
follow.
(1) Increased number and size of hyaline droplets in renal
proximal tubule cells of treated male rats
The abnormal accumulation of hyaline droplets is a necessary
but not sufficient feature of compounds that induce renal tubule
tumors through the G2u-g sequence of events and helps differentiate
them from chemicals that induce renal tubule tumors through ether
means.
(2) Accumulating protein in the hyaline droplets is C2u-g
Hyaline droplet accumulation is a nonspecific response to
protein overload in the renal tubule and need not be due to a~ -g
(e.g., as with chlorothalonil). Therefore, it is necessary to
demonstrate that a2u-g accumulation accounts for the hyaline
droplets found in the male rat.
(3) Aspects of the pathological sequence of lesions
associated with c*2u~g nephropathy are present.
Typical lesions include: single cell necrosis, exfoliation of
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epithelial cells into the proximal tubular lumen, formation of
granular casts, linear mineralization of papillary tubules, and
tubule hyperplasia. Some elements may not be visibly present if
the response is mild. Nevertheless, some endpoints should always
be observable.
B. Additional information useful for the analysis
If the preceding analysis (section IV-A) indicates that the
«2u-g process is a determinant in the observed renal effects,
then other types of information are reviewed to decide if the renal
effects are most likely due solely to the a2u-g-associated
phenomenon or whether this process in combination with other
potential carcinogenic processes is more likely. Many kinds of
information are available to assist in strengthening the
determination that chemically-induced a2(j-g accumulation appears to
be involved in the renal tumor response or that other processes
cannot be ruled out. Some of these findings are listed below; the
information should not be considered exhaustive.
(1) Hypothesis-testing data
The determination that the Q2u-g sequence is involved in the
renal tubule tumor response would be greatly enhanced by the
availability of specialized test results. Such information might
include: modification of the nephrotoxic response through
manipulation of sex hormones (e.g., androgens), a2u-g levels (e.g.,
a2(j-g administration to female rats), or use of the NBR rat. Other
information might include initiation-promotion studies comparing
males of the NBR strain with males of other rat strains.
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(2) Additional biochemical information
Certain in vivo and in vitro data help characterize a chemical
as one that would induce accumulation of a2u-g. Such information
might include: demonstration of reversible binding of the chemical
(or metabolites) to cz2u-g, demonstration of a reduction in the
lysosomal degradation of the a2u-g-complex, and disposition studies
demonstrating sex- and species-specific retention of the test
compound in the male rat kidney.
(3) Sustained cell division in proximal tubule of the male
rat.
Demonstration in the male rat of a sustained increase in cell
replication in the P2 segment of the renal tubule and a dose-
related increase in atypical hyperplasia of the renal tubule is
consistent with a conclusion that the cc2u-g process is operative,
especially if other laboratory animals were tested and did not show
similar responses. These endpoints may be non-specific for a2u~g-
inducers, however, since there are other renal carcinogens also
thought to affect the P2 segment of the renal tubule.
(4) Structure-jotivity relationships
Structure-acti\ i :~ •_• relationships for ch^i.i.~:als that induce a2u~
g accumulation in the :n-. ]o rat kidney are n^~ well defined,
although there appear to be dimensional reguirements to fit the
protein pocket, a reguirement for a degree of lipophilicity, and a
need for an electronegative atom in the molecule or its active
metabolite. Other structural features may suggest that a chemical
may also belong to a different class of suspected carcinogens.
(5) Covalent binding to macromolecules
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Some inducers of renal tubule cancer in rodents (e.g.,
nitrosamines) are known to bind covalently to DNA or other
macromolecules. Others do not appear to bind to DNA (e.g.,
isophorone) suggesting that such information may assist in
distinguishing different processes leading to renal cancer.
(6) Genotoxicity
Although renal tubule neoplasia associated with clearly
genotoxic chemicals is a well known response, information to date
supports a conclusion that a2u~<3 inducers are essentially
nongenotoxic and do not depend on direct genetic injury for the
production of tumors. Thus, information on potential genotoxicity
in a standard battery of short-term tests relevant to the
evaluation of potential carcinogenicity provides a possible device
for helping to distinguish between these processes.
(7) Nephrotoxicity
Although the presence of chronic progressive nephropathy (CPN)
in the aging male rat can complicate the analysis of other renal
lesions, if there is additional nephrotoxicity seen in the male rat
not attributable to either CPN or ct.2u~g accumulation, or if there
is a nephrotcxlc r;sponse in the female rat or the mouse, then the
possibility of other processes leading to renal cancer should be
considered.
(8) The availability of animal bioassay data in other
species-, sex- combinations.
The a2u-g-syndrome is a male rat-specific event. Positive
cancer responses in the renal tubule in female rats, mice of either
sex, or any other laboratory animal immediately suggest that the
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a2u-g syndrome, alone, is unable to account for the renal tubule
tumor response in the male rats.
The overall confidence in determining which of the three
categories applies in any given set of compound-induced renal
tubule tumors depends on the comprehensiveness of available data.
If these data all support a role for the involvement of chemically
induced a2u-g/ ^nere is a high degree of confidence in assuming that
the £2(J-g syndrome, alone, accounts for the renal tubule tumors.
In contrast, if information from adequate testing leads to serious
doubt about ct.2u~g involvement (see sections IV-A and IY-B) , there
is a high degree of confidence that other carcinogenic processes
account for the renal tumors. Sometimes, the information will
suggest that more than one carcinogenic process probably occurs; in
these cases, as a minimum, the criteria in support of a2u~9
involvement (section IV-A) are present, but there is also evidence
supporting involvement of other mechanisms (section IV-B).
Decisions on the applicability of the three categories can only be
made on a case-by-case basis, taking all of the information into
account. Whatever the finding, the risk assessor should clearly
delineate and thoroughly document the basis for any decisions made.
Once a decision on the applicability of the three categories
is made, it becomes possible to determine whether to use renal
tubule tumors in male rats to evaluate human hazard and to estimate
human cancer risk. In general, the following guidance applies,
recognizing that tumors occurring at other sites in laboratory
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animals administered compounds that induce a2u-g accumulation in the
male rat will be judged on their own merits.
1. For compounds producing renal tubule tumors in male rats
attributable solely to chemically-induced a2ij-g accumulation, the
renal neoplasms will not be used for human cancer hazard
identification or for dose-response extrapolations.
2. For compounds producing renal tubule tumors that do not appear
to be linked to the accumulation of a2u-g, those tumors will be used
both for human hazard identification and for quantitative risk
assessment.
3. For compounds producing renal tubule tumors in male rats
attributable to the a2u-g syndrome and to some other carcinogenic
process, recognizing that some portion of the renal tubule tumors
in the male rat remain relevant to hazard identification, a
preference is generally given for not basing the risk estimation on
the renal tubule tumors.
If there is enough information available to determine the
relative contribution of each process to the overall renal tubule
cancer response in male rats (e.g., from new mechanistic approaches
or hormone manipulation studies), this information may be used
accordingly both for hazard identification and dose-response
analysis. Clearly, case-by-case determinations are needed, and the
rationale for any position should be thoroughly presented.
V. Guidance for evaluating nephropathy as a toxic endpoint
If a compound induces o;2u-g accumulation in hyaline droplets,
the associated nephropathy in male rats is not used as an endpoint
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i
to determine noncancer (systemic) effects potentially occurring in
humans. Likewise, quantitative estimates of noncancer risk (e.g.,
reference doses and margin-of-exposure determinations) are based on
other endpoints wherever possible.
It should not be anticipated that a compound that produces
nephropathy in the male rat through the sequence of events
beginning with the accumulation of a2u-g will always be found to
induce renal tubule tumors in the male rat. The ability to detect
neoplasia depends on many features that may not be present in any
individual experiment, eg. sufficient dose to induce effect without
early deaths of the animals, competing toxicity from other moieties
not bound to a2lj-g, insufficient length of exposure or follovup, and
incomplete histopathology. Even in the absence of renal tubule
neoplasia in the male rat, if the sequence of lesions
characteristic of the a2u-g syndrome are present, the associated
nephropathy in the male rat does not contribute to determinations
of noncarcinogenic hazard or risk.
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