Background Findings that Confound Study Interpretation: Rodents, Dogs and Monkeys
Peter C. Mann, DVM; Diplomate, ACVP; Fellow, IATP
Senior Pathologist/Manager
Experimental Pathology Laboratories, Northwest
Seattle, WA, 98119
pmann@epl-inc.com
All the major species of laboratory animals commonly used in preclinical toxicology studies develop
changes that not the result of exposure to test-compounds but rather the result of natural ageing
process and the exposure to non-specific environmental agents. Rodents, which are generally
maintained behind barriers still develop some changes over time. Large animal species (dogs and
rodents) also have their own set of background findings.
Since the body has a limited number of responses to stimuli, the responses seen after administration of
novel compounds are often an exacerbation of the natural background responses. One of the major
tasks of the toxicologic pathologist is the separation of a treatment-induced lesion from a similar normal
background change.
One of the major tools used to sort out these differences is use of control group(s). By definition, a
control group consists of age-matched animals that are treated exactly the same (feeding, housing,
ventilation, etc.) as the treated groups, but which have not been administered compound. Therefore, it
can be concluded that any changes seen in the control group are not the result of treatment, but rather
a normal response to age and environment. Indeed, interpretation of the incidence and severity of
changes in treated groups compared to controls is one of the keystones of toxicologic pathology.
Generally, the control group is administered the vehicle that is used for drug delivery. In cases where the
vehicle itself might be expected cause changes, or where the procedure of administration (for example
subcutaneous injection) might cause a specific set of changes, there may be two control groups, one
with no vehicle, and a second with the vehicle to allow for finer discrimination of treatment-related
changes.
Another issue with the interpretation of background changes involves the use of thresholds as a criteria
for whether or not a change should be diagnosed. In an attempt to reduce the number of data points,
and make interpretation easier, a pathologist may choose an arbitrary threshold line, and only diagnose
those changes which are above (more severe) than the line, while those that are below the line (less
severe) are judged to be clearly background and therefore normal, and thus need not be diagnosed.
There are several weaknesses to this approach. First, the ability to differentiate normal from background
lesions is species and study specific – background changes in a one month rodent study are not the
same as those in a one month monkey or dog study, or even those in a 2 year rodent study. Thus proper
interpretation of background lesions requires experience on the part of the pathologist. Second, a
threshold is moving target – the same pathologist may examine two slides which appear objectively to
have the same set of changes, and yet declare one to be below the threshold (no diagnosis), while the
other gets a grade of minimal. Pathologists are (mainly) human, and may overlook a small focus which
would elevate a change to minimal, or their threshold may have changed over the course of study due
to the influence of having seen multiple animals with similar changes. This last effect, known as
diagnostic drift, can occur in any study, but is most prevalent in longer term studies that may take
several months to evaluate.
Since thresholds are inherently flawed, they should never be used to evaluate changes in suspected
target organs. In these cases, all changes should be diagnosed, and the resulting tables should be
evaluated to determine whether or not the change is treatment-related or background.
Chronic progressive nephropathy (CPN) is a classic example of a background change that may be
exacerbated by treatment, and which needs to be delineated from a treatment-related change. This
syndrome is a natural, age-related change in rodents (especially rats) in some strains may approach
100% at the end of a two-year study. Obviously, if the incidence is high and similar in both control and
treated group, one cannot use incidence alone to determine if the change is treatment-related. In these
cases, there may be an increase in the severity of the change in treated animals, which would help in the
interpretation of the changes. One would also like to see a dose-relationship to the increased incidence
and/or severity to aid in the interpretation.
CPN can also cause difficulties in interpretation in shorter term studies. As a syndrome, CPN consists of
several components: tubular basophilia (degeneration/regeneration), peritubular fibrosis, inflammatory
changes (primarily mononuclear), glomerular changes, tubular dilatation, and proteinaceous casts. The
diagnosis is simple when most or all of these changes are present, as would be likely in an older rat.
However, in a young rat, only the tubular degeneration/regeneration (generally considered to be the
primary change of CPN), or the mononuclear cell infiltration may be present. Since either of these
changes alone could be the result of primary renal toxicity, it is best to separate the components of CPN
and determine if any or all of them are the result of treatment. In longer term studies, where many of
the animals exhibit full-blown CPN, there is little to be gained by analyzing the component changes
separately.
One of the most common responses to injury in many organs is infiltration by mononuclear cells. These
small foci are commonly seen in the liver, prostate, kidney, and many other organs. There is often an
apparent increase in the incidence and/or severity of this change in treated animals when compared to
controls. This increase is often considered by the pathologist to be spurious – a reflection of the range of
response that might be seen as a background change. The difficulty arises when a reviewer notes a
small increase in incidence, and is concerned that this change represents a clear treatment-related
effect, while the study pathologist sees an small increase in incidence of a common background change.
The reviewer then asks for historical control data, which unfortunately often does not exist for nonneoplastic lesions in short term studies, or constitutes a small data set which does little to reassure.
Cardiomyopathy is another background change that may be exacerbated by treatment. The diagnosis
represents a group of changes in the heart which vary over time. In young rats, the changes include
degeneration of cardiomyocytes and mononuclear inflammatory cells. In older rats, these initial changes
are often gone, replaced by focal fibrotic changes. The diagnosis has been used for years; however it
may be more appropriate to diagnose the individual components, rather than use the diagnosis of
cardiomyopathy, since the latter term has specific connotations for our MD colleagues which are not
comparable to the changes in rodents (Keenan, 2010). Cardiomyopathy may be present in control
animals at an incidence of up to 100% (Jokinen, 2005); however the incidence in a particular study may
be lower, leading to an interpretation that this change may be treatment-related. Because of the limited
possible responses of the heart, primary cardiotoxicants may cause similar changes.
Dogs also have cardiovascular changes which may be either background or treatment-related. These
include vascular necrosis and polyarthritis (so-called Beagle Pain Syndrome (Snyder, 1995), as well as
myocardial necrosis (Greaves, 2000). Differentiating background from treatment related lesions is often
difficult (Clemo, 2003).
Eosinophilic droplets in the nasal epithelium of the nares represent another background change which
may be exacerbated by treatment. These bright eosinophilic deposits often fill the cytoplasm of affected
cells. They appear to be a non-specific response to a variety of nasal irritants. An increase in incidence
and severity may thus represent a secondary response to the irritating nature of either the compound or
vehicle, rather than a primary toxic response.
A few years ago, a mixed inflammatory change was noted in the alveoli of rats on toxicology studies
(Elwell, 1997). This apparently novel change was present in both control and treated animals. The cause
of the change remained unknown until 2012 (Henderson, 2012), when it was shown to be associated
with Pneumocystis carinii. While it is fascinating that this infectious agent can cause disease in
immunocompetent rats, it makes it less likely that the changes are the result of a primary toxic effect in
these rats.
Young male dogs and monkeys take several years to achieve sexual maturity. For this reason, the testes
of these species are immature at the time of termination. Because of the lack of normal
spermatogenesis in these immature testes, it is impossible to accurately assess the effects of
compounds on these young male animals, and it is inappropriate to make any diagnosis other than
“immature”. In addition, young male monkeys often have large areas of fibrous tissue in the testes,
where normal seminiferous tubules have not yet developed. This lack of normal development should
not be diagnosed as fibrosis.
In older rats, there is often severe degeneration of almost all the seminiferous tubules in one testis.
Since only one testis is affected, and the blood flow to each testis is similar, it has long been assumed
that this sort of change is spontaneous, and likely the result of prior trauma. However, there is recent
evidence that some compounds can cause a functional narrowing of the spermatic artery which results
in an infarct-like lesion in only one testis.
Cysts may be present in many organs, in organs including the thyroids, parathyroids, pituitary, and
thymus, these cysts are generally congenital and a result of variations in normal development (i.e.
thyroid cysts are often remnants of ultimobrancial bodies). These types of cysts are never treatmentrelated. Many recording systems do not grade cysts but only record them as present. Some pathologists
do not record these cysts at all.
Cysts in other organs, such as the kidney or ovaries, may be related to age, or may be the result of
primary or secondary toxicity. Careful evaluation of all such cysts and interpretation of tables will enable
the pathologist to separate background from treatment-related cysts.
Extramedullary hematopoiesis (EMH) is a common finding in rodents, especially in the rat spleen. Often
animals with ulcerated foot pads or tumors or systemic disease will have significant EMH. Careful
evaluation of the microscopic tables for the incidence and severity of EMH should help to differentiate
these individual animals from a true group effect.
With age, the thymus of rodents, dogs and monkeys undergoes a spontaneous involution.
Microscopically, this is often difficult to differentiate from primary lymphoid depletion, necrosis and
atrophy caused by immunosuppressive compounds. In addition, the thymus is one of the primary organs
affected by stress (Everds, 2013). It is very important to differentiate the secondary effects of stress on
the immune system from primary thymic toxicity from normal involution.
Assessment of the carcinogenic potential of a compound is one of the important aspects of pre-clinical
testing in laboratory species. When the incidence of tumors increases only slightly in treated animals,
the proper interpretation of the effect becomes more difficult. Statistical analysis often differentiates
between common and uncommon tumors. In addition, there are generally robust historical control
databases (both concurrent data from the lab where the study was conducted, as well as published
data) to help put the incidence and significance into proper prospective. In recent years, there has been
an apparent increase in the occurrence of tumors in very young rodents (in studies of 6 months or less).
These tumors have occurred in the kidney (Crabbs, 2013), where they have been shown to be familial, as
well as in the mammary gland and the lymphoreticular system (lymphomas). Because of the very low
incidence of neoplastic changes in young animals, even a single tumor in a high dose animal (with none
in the control) can be difficult to interpret.
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