Scientific and Regulatory Policy Committee

Toxicologic Pathology
2016, Vol. 44(2) 163-172

STP Best Practices for Evaluating Clinical ª The Author(s) 2016

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Pathology in Pharmaceutical DOI: 10.1177/0192623315624165
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Recovery Studies

Lindsay Tomlinson1, Lila Ramaiah2, Niraj K. Tripathi3,

Valerie G. Barlow4, Allison Vitsky5, Florence M. Poitout-Belissent6,
Denise I. Bounous7, and Daniela Ennulat8

Abstract
The Society of Toxicologic Pathology formed a working group in collaboration with the American Society for Veterinary Clinical
Pathology to provide recommendations for the appropriate inclusion of clinical pathology evaluation in recovery arms of nonclinical
toxicity studies but not on when to perform recovery studies. Evaluation of the recovery of clinical pathology findings is not
required routinely but provides useful information on risk assessment in nonclinical toxicity studies and is recommended when the
ability of the organ to recover is uncertain. The study design generally requires inclusion of concurrent controls to separate
procedure-related changes from test article–related changes, but return of clinical pathology values toward baseline may be suf-
ficient in some cases. Evaluation of either a select or full panel of standard hematology, coagulation, and serum and urine chemistry
biomarkers can be scientifically justified. It is also acceptable to redesignate dosing phase animals to the recovery phase or vice versa
to optimize data interpretation. Assessment of delayed toxicity during the recovery phase is not required but may be appropriate in
development programs with unique concerns. Evaluation of the recovery of clinical pathology data for vaccine development is
required and, for efficacy markers, is recommended if it furthers pharmacologic understanding.

Keywords
clinical pathology, toxicologic pathology, preclinical research and development, preclinical safety assessment/risk management

Introduction and Background
Previous literature has addressed best practices in study design
and evaluation of the recovery phases for nonclinical toxicity
studies (Perry et al. 2013; Pandher, Leach, and Burns-Naas
2012; Sewell et al. 2014). While clinical pathology was
mentioned in these articles, specific considerations for the
appropriate inclusion of clinical pathology evaluation were not
explored in detail. Standard clinical pathology biomarker
(alternatively known in general as ‘‘variable’’ and as ‘‘analyte’’
for a chemistry biomarker) evaluation is critical for the moni-
toring of potential toxicity in the clinic following characteriza-
tion in nonclinical studies, and it is also crucial to the
understanding of recovery from toxicity. Since regulations for
inclusion of clinical pathology in recovery arms of toxicity
studies are neither detailed nor stringent, an opportunity exists
for a review of the common practices in the pharmaceutical
industry and to make recommendations based on sound scien-
tific practices, with input from pathologists having diverse
backgrounds. The Scientific and Regulatory Policy Committee
of the Society of Toxicologic Pathology (STP) formed a work-
ing group in collaboration with the Clinical Pathology Interest
Group of STP and the Regulatory Affairs Committee of the
American Society for Veterinary Clinical Pathology (ASVCP)
to provide insight on the following points regarding inclusion
of clinical pathology evaluation in recovery studies: (1) appro-
priate comparisons for assessment of recovery of clinical
pathology, (2) considerations of tissue recovery, (3) selection
of recovery biomarkers based on dosing phase data, (4) consid-
erations for exploratory or newly approved biomarkers,
1
Pfizer Inc., Andover, Massachusetts, USA
2
Envigo (formerly Huntingdon Life Sciences), East Millstone, New Jersey, USA
3
Covance Inc., Madison, Wisconsin, USA
4
Valerie G. Barlow Consulting, LLC
5
Pfizer Inc., La Jolla, Massachusetts, USA
6
Charles River Laboratories, Senneville, Quebec, Canada
7
Bristol-Myers Squibb Company, Princeton, New Jersey, USA
8
GlaxoSmithKline, King of Prussia, Pennsylvania, USA
Corresponding Author:
Lindsay Tomlinson, Pfizer Inc., 1 Burtt Rd., Andover, MA 01810, USA.
Email: lindsay.tomlinson@pfizer.com
164 Toxicologic Pathology 44(2)

Table 1. Common Study Designs for Clinical Pathology Recovery Evaluation.a

Recovery
Dosing phase phase N/dose/ Length of Length of
Nb/dose/sex sex (N of dosing recovery
Species (N of groups) groups) phase phase Clinical pathology time points Clinical pathology evaluation
c
Mouse 10 to 20 (4) 5–10 (2 or 4) 2–13 weeks 2–4 weeks End of dosing + end of recovery Hematology and/or chemistry
Rat 10 (4) 5 (2 or 4) 2–26 weeks 2–13 weeks +Interim, end of dosing, and Hematology, coagulation,
+end of recovery chemistry, urinalysis, and/or
biomarkers or noncore
biomarkers
Dog/monkey 3–4 (4) 2–4 (2 or 4) 2–26 weeks 2–13 weeks Pretreatment(s), +interim(s) Hematology, coagulation,
and end of dosing, +interim chemistry, urinalysis, and/or
recovery and end of recovery biomarkers or noncore
biomarkers
a
Regulated good laboratory practice studies form the basis of this experience.
b
N ¼ number.
c
Different cohorts will be evaluated at end of dosing and end of recovery.

(5) evaluation of delayed toxicity, (6) biomarker recovery in phase as recommended by Weingand et al. (1996) and then mon-
vaccine studies, and (6) recovery of efficacy markers. itored at a frequency that allows assessment of changes over time
(EMA Committee of Human Medicinal Products/Safety Working
Party 2010). If there is a need for single-dose toxicity studies,
Current Regulatory Guidelines hematology and clinical chemistry data should be evaluated
*24 hr after a single administration, with further evaluations
Regulatory guidelines from organizations and agencies responsible
conducted 2 weeks later to assess for delayed toxicity and/or
for the investigation of the safety and/or efficacy of chemicals or
recovery (EMA Committee of Human Medicinal Products 2010).
human health products, such as the International Conference of
The World Health Organization (WHO) guidelines on vac-
Harmonization (ICH), U.S. Food and Drug Administration (FDA),
cines (Guidelines on Nonclinical Evaluation of Vaccines; WHO
Environmental Protection Agency (EPA), European Medicines
2005) are an exception as they provide detailed recommendations
Agency (EMA), Organisation for Economic Cooperation and
for the evaluation of findings during the recovery phase. WHO
Development (OECD), and the Japanese Ministry of Health,
recommends that the pivotal supporting nonclinical study should
Labor, and Welfare, provide specific recommendations for the
include additional treatment groups, which will be evaluated at
inclusion of body weight, food/water consumption, hematologic
later time points after vaccination to allow evaluation of the rever-
and clinical biochemistry measurements, and morphologic pathol-
sibility of adverse effects observed during the dosing phase and to
ogy investigations in nonclinical toxicity studies. Guidelines for
screen for potential delayed adverse effects. WHO indicates that
recovery evaluation have been recently reviewed (Pandher, Leach,
hematology and serum chemistry evaluation should be consid-
and Burns-Naas 2012; Perry et al. 2013).
ered within 1 to 3 days following the first and last dose adminis-
Few guidelines for pharmaceutical development provide
tration and at the end of the recovery phase (2 weeks or more
detailed recommendations regarding clinical pathology evalua-
following the last dose; WHO 2005). At a minimum, biomarkers
tion in the recovery phase of nonclinical regulatory toxicology
to be evaluated include an evaluation of relative and absolute dif-
studies. Recommendations for the inclusion of a recovery
ferential white blood cell counts, albumin/globulin ratio, enzyme
phase state that at least one nonclinical study over the course
activities (specific enzymes not detailed in the guideline), and
of the development of a compound should incorporate a recov-
electrolyte concentrations. In some cases, assessment of coagula-
ery phase at the end of the study to determine the reversibility
tion times and fibrinogen concentration, urinalysis, and serum
or potential worsening of test article–related effects; guidelines
immunoglobulin classes are recommended.
specify that recovery groups should be monitored until reversi-
bility is demonstrated but full recovery is not necessary (FDA
2003; OECD 2008, 2009; ICH 2011; EPA 2001). However, an
alternative to including a recovery phase is using scientific jus-
Current Pharmaceutical Practices
tification as to whether a toxicologic effect with potential Common practices for the evaluation of clinical pathology dur-
adverse clinical impact is reversible. A nondosing period is ing the recovery phase of nonclinical toxicity studies were
warranted in a chronic study if there is severe toxicity at rele- compiled based on evaluation of the literature and the experi-
vant clinical exposures or if toxicity is only detectable at an ence of the working group and are presented in Table 1. The
advanced stage of the pathophysiology in humans. number of recovery groups may be similar to the number of
A few of the EMA guidelines indicate that in repeat-dose stud- dosing phase groups or limited to control and high-dose groups.
ies, clinical pathology should be evaluated during the dosing The study design relative to inclusion of recovery groups may
Tomlinson et al.
be influenced by many factors including the availability of data
from previous studies, company preference, or an attempt to
minimize animal usage. The number of animals designated for
the recovery phase is usually smaller than that used during the
dosing phase (e.g., one-third to two-thirds of that in the dosing
phase), although this could vary depending on the anticipated
incidence of findings. Recommendations in this article are not
based on a specific study design because the number of recov-
ery groups and the number of animals evaluated per recovery
group is unique to each test article. However, it is strongly rec-
ommended that the clinical pathology recovery assessment
strategy be determined in consultation with a clinical patholo-
gist and ideally with the one who will be interpreting the data.
Standard clinical pathology biomarkers (Tomlinson et al.
2013) are generally evaluated from individual recovery ani-
mals at the end of the dosing phase in most laboratory species
(e.g., rats, dogs, and monkeys) other than mice as this helps
with the assessment of reversibility in individual animals. In
some studies, changes in individual clinical pathology biomar-
kers may help to determine which animals are to be retained for
evaluation during the recovery. Particularly in rodents, evalua-
tion of different cohorts at the end of the dosing and recovery
phases may be required due to blood and sample volume lim-
itations, thus preventing evaluation of recovery in the same
individual animals. Repeated collections are rarely possible
in mice as blood collection is usually a terminal procedure in
this species. The length of the recovery phase is generally
determined by the half-life of the test article or the nature of the
test article–related findings. In most cases, all clinical pathol-
ogy biomarkers that were assessed during the dosing phase are
evaluated during the recovery phase. However, in the recovery
phase, some investigators may opt to evaluate only the subset
of biomarkers that were altered by target organ toxicity and/or
test article pharmacology during the dosing phase. This deci-
sion can be determined on a case-by-case basis as long as there
is scientific justification for it. For appropriate data compari-
sons to be made, blood sampling and analysis conditions (site
of collection, fasting, anesthesia, and type of tube and analysis)
should be the same for the dosing and recovery phases.
A large global retrospective analysis detailing general prac-
tices for use of recovery groups (Sewell et al. 2014) provided
recommendations for minimizing recovery animals where
scientifically justified, including avoiding recovery phases in
first in human (FIH)-enabling studies. A broad review of the
literature suggested that clinical pathology is most often incor-
porated in the recovery groups to evaluate reversibility of
organ dysfunction (such as renal, hematopoietic, immune, and
endocrine systems), with fewer reports incorporating clinical
pathology to monitor tissue injury (Abraham, Gottschalk, and
Ungemach 2005; Boorman et al. 1982; Henzen et al. 2000;
Derelanko et al. 1985; Lefebvre et al. 1984; Rouse et al.
2011; Streck and Lockwood 1979; Terse et al. 2011).
A retrospective analysis of studies performed over the past 6
years at a large U.S.-based contract research organization indi-
cated that recovery groups were included in *25% of studies.
In most cases, the full clinical pathology profile was routinely
165
included in the recovery phase. In a few instances, selected
clinical pathology biomarkers were included if a test article–
related effect was observed in these test(s) during the dosing
phase.
Appropriate Comparisons for Assessment
of Recovery in Clinical Pathology
Nonclinical study design recommendations by the FDA to sup-
port clinical trials do not specify which comparisons should be
made for evaluation of reversibility. Reference values or poten-
tial comparators in toxicology studies are often obtained from 1
or more of 3 sources: a concurrent control group not exposed to
the test article, baseline (alternatively known as ‘‘pretest’’ or
‘‘prestudy’’) values compiled from all animals prior to dosing,
or historical reference intervals collected from defined control
and/or predose animals. Importantly, utilizing historical control
values or ‘‘reference ranges/intervals’’ can result in misinter-
pretation of data, unless there is strict partitioning for animals
of the same strain; age; sex; provider; comparable routes of
administration; and vehicles and exclusion of animals with
individual conditions, histories, and/or treatments (Hall 1997;
National Committee for Clinical Laboratory Standards 1995;
James 1993). The practice of strict partitioning prevents highly
variable historical data which would be derived if control
groups from nonclinical studies were given a variety of vehi-
cles (Figure 1). The ASVCP has also developed guidelines for
establishing reference intervals (http://www.asvcp.org/pubs/
pdf/RI%20Guidelines%20For%20ASVCP%20website.pdf).
In rodent studies, a concurrent control group is considered to
be necessary for the interpretation of data (Weingand et al. 1996;
Leissing, Izzo, and Sargent 1985; James 1993). Using concur-
rent controls is recommended because there is less interanimal
variation than with the use of historical data, it eliminates con-
cerns with collecting baseline data from the same animals, and
the animals are the same age at the end of the study. In accor-
dance with suggested animal numbers (ICH 2009), rodent stud-
ies commonly incorporate larger numbers of genetically similar
animals per group than nonrodent studies, resulting in lower bio-
logical variance. Due to the small differences between individ-
ual animals, a control group of 5 animals/gender is sufficient
to assess recovery for the standard biomarkers. Because of low
blood volumes, particularly in young rodents, baseline blood
collection can negatively impact results and interfere with study
interpretation or overall animal health. Satellite groups for other
purposes such as toxicokinetics are often included to avoid
impact of blood collection procedures on recovery animals. Due
to the shorter life span of rodents, the periadolescent age that
rodent studies typically start, and potential age-related effects
on certain analytes (e.g., age-related decreases in serum phos-
phorus; Wolford et al. 1987), inclusion of a concurrent control
group ensures comparison to appropriate age-matched controls
(Pandher, Leach, and Burns-Naas 2012).
Studies in nonrodent species (dogs and nonhuman primates)
generally use smaller numbers of more genetically diverse ani-
mals with higher interanimal variation but lower intraanimal
166 Toxicologic Pathology 44(2)

Figure 1. Variable alanine aminotransferase values in control male Han-Wistar rats given a variety of vehicles.

variation; therefore, evaluation of baseline values is recom- phases (Leissing, Izzo, and Sargent 1985; Boone et al. 2005;
mended to better understand the biological variance and aid James 1993). However, the study purpose and/or complexity
in the interpretation of results during the dosing and recovery of the study design may dictate the need for concurrent controls
Tomlinson et al.
in the recovery phase. For short-term studies with low animal
numbers and limited blood collection or other evaluations dur-
ing the dosing phase, low intra-animal variation may make it
preferable to monitor the return toward baseline of potential
test article–related changes in individual animals. However,
as the study design becomes more complicated with additional
pharmacokinetic, pharmacodynamic, antidrug antibody, immu-
nophenotyping or other evaluations, separation of procedure-
related changes from test article–related changes may not be
possible without a concurrent control group. Nonhuman pri-
mates are particularly susceptible to impact red cell mass
because of the blood volume requirements for additional biomar-
kers that can be more easily translated to the clinic than in other
nonrodent species.
Considerations of Tissue Recovery
A crucial component of evaluating the reversibility of toxicity
is the assessment of biomarkers indicative of structural organ
damage and/or alteration of organ function. The standard
recommended evaluations during the dosing phase and/or
recovery phase include hematology, coagulation, clinical
chemistry, and urinalysis panels to assess alterations in major
organ systems (Tomlinson et al. 2013).
For the majority of test article–related changes in specific
biomarkers, it is useful to monitor the return of those results
back toward baseline levels, particularly when the recovery
of clinical pathology findings is not well documented for a
given test article. However, with some test article–related
changes, there is no realistic potential that a biomarker will
return to baseline, and thus measurement of the relevant bio-
markers would not be warranted. An example is a permanent
change in an organ such as the endocrine pancreas where the
utility of evaluation of functional markers (e.g., insulin and glu-
cagon) during the recovery phase is questionable in the pres-
ence of advanced nonrecoverable damage to the islets. Perry
et al. (2013) provided examples of anatomic pathology findings
that are not expected to recover; one example is injury to per-
manent or terminally differentiated tissues, such as cardiac
muscle. As cardiomyocytes are considered postmitotic, there
is no reasonable expectation that regeneration will occur. Thus,
Perry et al. (2013) contend that recovery of the microscopic
lesions should not be evaluated in such cases. With cardiac
muscle injury, common analytes of assessment may include
aspartate aminotransferase (AST) and creatine kinase (CK)
activities and cardiac troponin I (cTnI). These analytes may
be used to monitor cessation of active degeneration, but they
are not indicative of restored functional capacity of the heart
since irreversible cardiac injury resolves with fibrosis. Simi-
larly, evaluating analytes should be carefully considered when
stable tissues (quiescent with the ability to regenerate with
appropriate stimuli, such as renal tubular epithelial cells or
hepatocytes) have severe enough structural injury that tissue
architecture is not expected to be restored. For example, with
severe liver injury resulting in hepatic fibrosis that bridges and
dissects liver lobules, recovery of hepatic injury marker
167
changes (e.g., alanine aminotransferase and AST activities)
would not reflect a full recovery of liver structure or function.
When tissue injury is expected to fully recover and can be
easily monitored in a clinical setting, whether associated with
microscopic changes or not, assessment of the recovery of bio-
markers associated with that tissue injury may not be required.
This decision should be based on the information available for
the test article and/or pharmacologic mechanism. If the time
course of damage and recovery is well understood for a test
article and its related toxicity, then monitoring associated bio-
markers after the recovery phase adds no additional informa-
tion and is not required. For example, altered biomarkers
from labile tissues (those that are continuously dividing), such
as hematopoietic cells in the bone marrow and surface epithe-
lium of the gastrointestinal tract, are expected to return to base-
line/control levels. In these cases, the timing of the return
toward baseline/control levels may be more critical than
demonstration of full recovery. In agreement with Perry et al.
(2013), when loss of architecture occurs in a labile tissue (such
as bone marrow) or there is injury to a stable tissue (such as
liver) but normal architecture is retained, there is more uncer-
tainty about the ability of the tissue to recover. In those cases,
morphologic recovery should be evaluated along with appro-
priate associated biomarkers, with consideration of the half-
life of the test article and biomarkers and length of the recovery
phase. For example, monitoring liver enzyme activity can pro-
vide information regarding ongoing liver injury, as well as
timing of recovery. This could be particularly helpful when
evaluating either small molecules or biologics with long termi-
nal half-lives (several days to weeks).
The concurrent recovery of clinical pathology changes and
clinical observations may help to distinguish changes that are
secondary to poor clinical condition (e.g., decreased body
weight or stress-related signs) from primary test article–related
effects. For example, the return toward baseline/control levels of
biomarkers associated with dehydration (e.g., urea and creati-
nine) and decreased food consumption (e.g., ALP, albumin, tri-
glycerides, and effects on hematopoietic biomarkers such as
reticulocytes) in the recovery phase should be correlated to the
clinical signs of the recovering animals. If clinical pathology
changes in the dosing phase are clearly identified as secondary
or have been previously characterized in the program as second-
ary, evaluation at the end of the recovery phase is not required.
Selection of Recovery Biomarkers Based on
Dosing Phase Data
In general, options for clinical pathology assessment following
a recovery phase can include evaluation of the full panel,
portions of the panel such as hematology but not clinical chem-
istry, or only affected biomarkers. A prescriptive recommenda-
tion cannot be made because there are aspects, such as
biomarker storage stability, that affect the ability to delay
assessment and thus, decision making on the assessment. An
abbreviated panel can be considered if there are only changes
in a portion of the clinical pathology panel, such as fibrinogen.
168
If a decrease in fibrinogen is the only change at the end of the
dosing phase, the decision could be to evaluate the coagulation
panel (e.g., prothrombin time [PT], activated partial thrombo-
plastin time [APTT], and fibrinogen) or fibrinogen alone dur-
ing the recovery phase. In the latter case, the individual
laboratory or sponsor should consider logistical considerations
such as computer software templates to determine if there is an
appropriate cost–benefit scenario to run one test instead of a
typical panel of biomarkers. However, each laboratory has to
weigh these considerations against the risk of creating unneces-
sary data versus the risk of producing a limited data set that
cannot be interpreted properly. For example, if there are
increases in fibrinogen alone at the end of the dosing phase,
evaluation of related inflammatory parameters in hematology
and serum chemistry panels should also be considered. Consul-
tation with a clinical pathologist is recommended during this
decision-making process.
There is limited utility in evaluating biomarkers after a
recovery phase when there were no test article–related altera-
tions at the end of the dosing phase. However, if clinical pathol-
ogy results from the dosing phase are not available in time for
interpretation and if waiting for evaluation compromises sam-
ple quality, then it may be preferable to routinely collect clin-
ical pathology samples and/or evaluate them at the end of a
recovery phase. Also, if there are specific concerns of potential
alterations due to test article exposure during the recovery
phase because of a long half-life, clinical pathology evaluation
may be warranted.
Assessment of clinical pathology during the recovery phase
should also be considered when test article–related clinical
pathology changes indicate a functional effect that is not expected
to have morphologic correlates or when clinical pathology
changes are more sensitive predictors of toxicity than morpholo-
gic ones. For example, a decreased reticulocyte count may indi-
cate injury to the bone marrow that is not yet apparent
histologically or has not yet impacted red blood cell (RBC) mass
because of the long life span of RBCs. If damage to the marrow is
severe enough in a short-term study, the reticulocyte count may
not improve or may decrease further during the recovery phase,
and RBC mass may be decreased at the end of the recovery phase
even though it was unchanged at the end of the dosing phase.
Interpretation of data can be challenging if appropriate time
points and animals are not evaluated. In rodent studies, suffi-
cient blood volume may not be available to sample every animal
at every desired time point. Special attention must be paid to
how animals are bled during the dosing phase and during the
recovery phase to minimize preanalytic variation and optimize
interpretation. For example, multiple collections during a study
(interim, end of dosing, and/or end of recovery) would enable
assessment at multiple time points in a single rat and correlation
with histopathology data for animals terminated at the end of
dosing or recovery phases. Bridging of doses between studies
can facilitate comparisons of clinical pathology and histopathol-
ogy data across studies; by utilizing data from shorter term stud-
ies, it is possible to minimize blood collection during the dosing
phase of longer term studies, allowing collection of appropriate
Toxicologic Pathology 44(2)
time points to interpret the recovery phase. For example, when
only two time points can be evaluated per animal, it is most ben-
eficial to collect data from end of dosing and end of recovery.
Animals from the control and selected dose group(s) are usu-
ally assigned to a cohort recovery group at the onset of the study.
However, under certain circumstances, the assignment of ani-
mals may be altered to include animals that have been identified
as having test article–related clinical pathology findings to
ensure that representative changes are present in the recovery
group and subsequently analyzed during the recovery phase. For
example, if 5 of the 10 animals in the high-dose group exhibit
marked hematologic changes, but none are assigned to the
recovery group, then reassignment may be considered so that
recovery of the finding can be assessed. However, if animals
assigned to the recovery phase have critical levels of a biomar-
ker (e.g., severe life-threatening thrombocytopenia or neutrope-
nia), it might be advisable to terminate them at the end of the
dosing phase. Similarly, if all animals with an increase in an
injury biomarker are in the recovery group, it may be impossi-
ble to determine the histologic nature of the lesion present at
the end of dosing without reassigning one or more animals to
the end of dosing phase. Such reassignments are only recom-
mended if they will make the data set more interpretable and
are more likely to be utilized in studies with large animals
rather than in rodents due to the smaller group size. A reason-
able justification of any reassignment should be well documen-
ted in a study amendment. Reassignment is not recommended
for animals with minor changes and may not be necessary for
animals with changes that are expected to fully recover. Ensur-
ing and documenting that appropriate animals are part of the
recovery group to allow adequate assessment of recovery is the
responsibility of the study director.
Considerations for Exploratory or Newly
Approved Biomarkers
Standard clinical pathology biomarkers have been used for
decades and their performance features (sensitivity, specificity,
kinetics), assay formats, and caveats are well understood. This
is not necessarily the case with novel safety biomarkers. Novel
exploratory safety or nontraditional biomarkers should be used
within a clearly defined context. The goals, anticipated out-
comes, and reference standard or comparators such as the
associated histopathology or functional change, should be iden-
tified prior to use in a study. The interpretation of the novel bio-
markers should be based on these predefined endpoints. In
some instances, very little is known about the biological role,
sensitivity, kinetics, and species differences of novel biomar-
kers. The assays themselves are often at the exploratory stage
of development and characterized as ‘‘fit for purpose’’ (Lee
et al. 2006; Wagner 2008). Gathering data during the recovery
phase will enable a progressive understanding of the candidate
biomarker, as interpretation of changes in standard clinical
pathology biomarkers and other study data can be used to put
information on the novel marker biology in health and disease
into context.
Tomlinson et al.
In 2011, the FDA accepted the qualification of cTnI as an
indicator of the presence and extent of cardiac structural dam-
age in dogs and rats based on data from studies of drugs with
known cardiotoxicity or drugs that have shown evidence of car-
diac damage when previously tested (http://www.fda.gov/
downloads/Drugs/DevelopmentApprovalProcess/DrugDeve
lopmentToolsQualificationProgram/UCM294644.pdf). The
intent of using cTnI as a biomarker of cardiac toxicity is to bet-
ter define the lowest cardiotoxic dose in nonclinical studies of
test articles known to induce structural cardiac damage and to
help choose starting doses in clinical studies of those test arti-
cles; there is no intent to use cTnI as a general screening marker
to detect unexpected cardiac toxicity. CTnI often peaks 2 to 6
hr after cardiac injury in nonclinical studies, depending on the
duration and extent of cardiac injury (Clements et al. 2010;
O’Brien 2008). Therefore, cTnI should be evaluated taking the
short circulating half-life and the timing of the peak concentra-
tion of the drug into account, usually within 24 hr after drug
administration. An increase in cTnI values, however, should
be carefully evaluated. As in people, obtaining serial samples
might help to evaluate the relevance of these changes in noncli-
nical studies. Given the kinetics and short circulating half-life
of cTnI, measurement during a recovery phase may not be war-
ranted, but the half-life of the test article must also be consid-
ered. A negative cTnI result does not indicate an absence of
structural cardiac damage.
Beginning in 2010, FDA accepted several qualification sub-
missions for 9 novel urinary biomarkers including kidney
injury molecule-1 (Vaidya et al. 2010), clusterin, cystatin C,
and renal papillary antigen-1 (Dieterle et al. 2010) that demon-
strated improvement in the detection of acute drug-induced
kidney injury in rats (Harpur et al. 2011). Subsequently, rat
urinary osteopontin and neutrophil gelatinase-associated lipo-
calin (NGAL) have received regulatory endorsement in the
form of a Letter of Support signed by Janet Woodcock on
August 20, 2014. Most of the data supporting the use of these
biomarkers were generated in 4- to 15-day studies, and recov-
ery was not evaluated. Even though these renal biomarkers are
accepted, there is still limited information about the kinetics,
sensitivity, specificity, recovery, or assay interferences for
them. The widespread use of these novel urinary biomarkers
in rodent nonclinical studies, including their use to evaluate
recovery of the target organ toxicity, will enable progressive
qualification and further understanding of the biology unique
to these markers.
Immunotoxicity findings (excluding immune-related find-
ings secondary to stress) may be observed as intended pharma-
cological effects or as unexpected (off-target) effects of test
articles. It is recommended that immunotoxicity findings
undergo further evaluation either in follow-up studies, as
described in the FDA guidance on immunotoxicology evalua-
tion of investigational new drug (FDA Center for Devices and
Radiological Health: U.S. Department of Health and Human
Services 1999), or during the recovery phase of toxicity studies.
For example, in the case of a lymphocyte-depleting antibody,
thorough immunophenotypic evaluation of lymphocyte subsets
169
and the kinetics of repopulation is essential for characterization
of the pharmacologic activity of the test article, identification
of potential adverse events, and determination of the most
appropriate clinical administration regimen. However, the
addition of recovery phases in order to assess immunogenicity
is not required as per ICH harmonized tripartite guideline, pre-
clinical safety evaluation of biotechnology-derived pharma-
ceuticals S6 (R1).
Evaluation of Delayed Toxicity
With the exception of nonclinical studies supporting microdos-
ing in the clinic, there is no regulatory requirement for the eva-
luation of delayed toxicity in nonclinical studies. However, in
nonclinical drug development, the addition of recovery groups
should be considered when there is a specific concern for test
article–related findings that could develop after the dosing
phase. For example, for test articles with known class liabilities
caused by effects on early hematopoietic or spermatogenic pro-
genitors, a recovery phase may be used to monitor changes that
may not manifest within the course of an FIH-enabling study.
Data from recovery groups may allow decisions on the design
of clinical dosing as well as subsequent nonclinical studies. In
the case of biopharmaceuticals, recovery groups are commonly
used to monitor for the potential of a test article to produce
delayed toxicity during the off-dose phase due to either pro-
longed exposure or extended pharmacodynamic effects of the
test article, although this is not required per ICH S6 (R1).
Although most of the concerns for delayed toxicity relate to
morphologic changes, clinical pathology data can aid in the
evaluation of target organ toxicities through identification of
effects on organ function that might not be evident morpholo-
gically. For example, a compound affecting lymphoid stem
cells in bone marrow could have delayed effects on peripheral
lymphoid subpopulations due to the progressive depletion of
circulating lymphocytes with a long half-life. Similarly, as
T-cell maturation occurs in the thymus, a test article causing
concurrent hematopoietic and thymic toxicity might exhibit a
delayed or more severe effect on peripheral T-cell counts than
a compound causing thymic injury alone due to the combined
effects of senescence of mature T cells and decreased bone
marrow lymphopoiesis. Finally, in the case of a nephrotoxicant
with proximal tubular injury, concurrent urinalysis biomarkers,
including evaluation of protein or albumin excretion with his-
topathology, could support demonstration of reversibility, par-
ticularly if there is histological evidence of tubular basophilia
in recovery animals. As tubular basophilia can reflect either a
regenerative response or a toxic change (Seely and Frazier
2015), recovery of urinalysis biomarkers can aid in the discrim-
ination between regenerative and adverse tubular changes.
Biomarker Recovery in Vaccine Studies
As described earlier, regulatory guidelines for the recovery
of clinical pathology biomarkers in the evaluation of vac-
cines are more detailed than for other areas of drug
170
development. Vaccine programs are unique since a dose–
response evaluation is not required as part of the basic toxi-
city assessment, and maximum tolerable doses do not need
to be determined. As vaccines are given episodically rather
than daily, the dosing intervals may be based on the kinetics
of the primary and secondary antibody responses observed
in the animal studies.
Vaccine administration causes acute phase protein increases
that will usually be at their peak 24 to 48 hr postinjection.
Therefore, only a mild, if any, change in acute phase proteins
should be observed during or at the end of the recovery phase.
While residual chronic inflammation is often described after
the 2-week off-dose phase and is reflective of an expected tis-
sue response, some recovery of the inflammation is expected
during that time. Unless the immunological and inflammatory
response to the vaccine is severe and prolonged, it is not con-
sidered to be adverse.
Extensive recommendations for vaccine evaluation have
recently been published and are being readily adopted by com-
panies developing vaccines (Green and Al-Humadi 2013).
These recommendations provide a larger and updated list of
biomarkers to consider during vaccine studies. The clinical
pathology biomarkers recommended for evaluation at all time
points include a complete blood cell count, a serum chemistry
panel, a coagulation panel including fibrinogen concentration,
and the measurement of acute phase proteins. The recom-
mended acute phase proteins are C-reactive protein (CRP,
which is the primary acute phase protein in rabbits, monkeys,
and humans) and a 2-macroglobulin and a 1-acidic glycopro-
tein in rodents (Green and Al-Humadi 2013) or more specifi-
cally in rats (Cray, Zaias, and Altman 2009). This STP/
ASVCP working group concurs with the recommendations but
also recommends CRP in dogs and serum amyloid A and
haptoglobin in mice (Cray, Zaias, and Altman 2009). When
specific acute phase proteins are measured, serum electrophor-
esis analysis is not required. The measurement of CK activity,
an analyte with a short half-life, is useful for the evaluation of
the recovery from muscle injury, particularly in dogs (Tomlin-
son et al. 2013; Vassallo et al. 2009). Since vaccine administra-
tion may cause local muscular degeneration, the evaluation of
CK activity or other biomarkers of skeletal muscle injury such
as skeletal troponin I (Vassallo et al. 2009), dependent on the
species being evaluated, is also recommended during the off-
dose phase of nonclinical vaccine studies.
Recovery of Efficacy Markers
Efficacy markers enable monitoring of desired pharmacologic
effects in both nonclinical studies and clinical trials and may be
useful to evaluate in a recovery phase to understand persistence
of these pharmacologic effects, particularly for biologic or
large-molecule test articles that have long half-lives. For small
molecules with short half-lives, efficacy is likely to decrease
quickly following cessation of dosing, and evaluation of recov-
ery for efficacy markers induced by these test articles is gener-
ally unnecessary.
Toxicologic Pathology 44(2)
A primary objective for the use of efficacy markers in non-
clinical toxicity studies is to identify the relationship between
intended pharmacologic effects and off-target or adverse find-
ings. For example, comparison of the peak exposure for desired
pharmacology with the exposure level where adverse findings
occur can be used to define the relationship between on- and
off-target effects. Subsequently, efficacy markers or clinical
pathology biomarkers related to either desired pharmacology
or target organ toxicity may be evaluated during a recovery
phase in conjunction with histopathology to define the relation-
ship between various findings or to inform the risk assessment
of both intended and unintended findings. Utilizing the desired
pharmacology, half-life, mode of action, and associated on- or
off-target effects will optimize the design for evaluation of effi-
cacy markers during the recovery phase.
The biological time course of a pharmacologic effect often
determines duration of recovery. For example, increased glu-
cose production due to treatment with a glucagon analog
quickly returns to normal levels after test article exposure
diminishes, whereas excessive production of RBCs caused by
erythropoiesis-stimulating agents or an excessive number of
leukocytes produced as a result of bone marrow stimulation
by granulocyte-colony stimulating factor reverse slowly because
the target cell populations may decrease slowly over time. In
cases where cell populations are expected to change slowly or
where long-lasting effects are expected, demonstration of com-
plete recovery to baseline may not be necessary. However, fre-
quent monitoring of efficacy markers can help to define the
length of recovery required in some cases, such as for biologic
test articles with longer half-lives. It may not be necessary or
beneficial to assess a full panel of clinical pathology biomarkers
if only intended pharmacology is being monitored.
Besides persistence of a pharmacologic effect, the need for
recovery assessment may depend on the organ involved and the
mechanism of the effect. With some immunomodulators, pro-
longed decreases in B- or T-lymphocyte populations can persist
without clinical or other hematologic manifestations. In rare
cases, adverse events can occur during the recovery phase due
to immunosuppression or unintended immunostimulation. If
such events are anticipated, close monitoring of efficacy mar-
kers, traditional clinical pathology biomarkers, and clinical
observations may be warranted throughout the dosing and
recovery phases. Also, with certain classes of test articles that
are known to have long-lasting effects (e.g., some bisphospho-
nates or endocrine modulators), slowly progressive or delayed
pharmacologic effects can occur and monitoring may be
desired during the recovery phase.
Finally, it is recommended that for the evaluation of the
recovery of efficacy markers, the pharmacologic effect should
be present in animals designated for recovery. This is of partic-
ular concern in studies with biologic test articles that induce
immunogenicity in the test species. Depending on the level
of development and the effects on test article exposure of
antitest article antibodies, not all animals will have similar
pharmacologic effects that can be monitored during the recov-
ery phase.
Tomlinson et al.
Conclusions
Regulatory guidelines provide the flexibility required for
adapting the recovery phase to the needs of the program, but
offer limited direction for the evaluation of clinical pathology
in the recovery phase. The mandate of this STP/ASVCP work-
ing group was to consider the assessment of and to provide con-
siderations and recommendations on the appropriate inclusion
of clinical pathology biomarkers in the recovery phase of non-
clinical toxicology studies.
The following recommendations represent the ‘‘best prac-
tice’’ for appropriate evaluation of clinical pathology biomar-
kers in the recovery phase of nonclinical toxicity studies.
(1) The clinical pathology recovery assessment strategy should
be determined after consultation with a clinical pathologist,
ideally the one who will be interpreting the data.
(2) Data from concurrent controls are recommended for
assessment of recovery data in rodent studies and com-
plicated nonrodent studies but in simple short-term
(<28 days) nonrodent studies, comparisons of recovery
data in test article–treated animals to baseline may be
adequate.
(3) Recovery of biomarkers of toxicity should be assessed
when the timing or ability of the organ to recover is of
concern (e.g., loss of architecture in a labile tissue or
injury to a stable tissue while normal architecture is
retained).
(4) An evaluation of biomarkers is not required after the
recovery phase if an associated group of biomarkers is
unchanged after the dosing phase, the recovery of test
article–related clinical pathology changes are well under-
stood, there is no expectation of recovery, the changes are
secondary effects, and/or the changes have no relevance
to recovery.
(5) Assessment of clinical pathology during the recovery
phase should be considered when clinical pathology
changes indicate a functional effect with no morphologic
correlates or when clinical pathology changes are more
sensitive predictors of toxicity than morphologic changes.
(6) Reassignments of dosing and recovery phase animals are
only recommended if they will make the data set more
interpretable.
(7) Evaluation of clinical pathology biomarkers of delayed
toxicity should be considered on a case-by-case basis in
development programs with specific concerns for delayed
toxicity that can be monitored using clinical pathology.
(8) In addition to the guidelines for clinical pathology assess-
ment of recovery from vaccine administration, evaluation
of acute phase proteins (CRP in dogs and monkeys, a 2-
macroglobulin or a 1-acidic glycoprotein in rats, and
serum amyloid A or haptoglobin in mice) and biomarkers
of skeletal muscle injury is recommended.
(9) Evaluation of novel exploratory or nontraditional clinical
pathology biomarkers during the recovery phase should
be used within a clearly defined context.
171
(10) For the evaluation of recovery of efficacy markers, the
pharmacologic effect should be present in animals
intended for the recovery phase.
These recommendations are intended to complement rather
than replace recommendations currently available in the litera-
ture and to provide additional clarification for the clinical
pathology evaluation in the recovery phase of nonclinical toxi-
city studies.
Acknowledgments
The working group acknowledges the input from members of STP and
ASVCP.
Author Contributions
Authors contributed to conception or design (LT, DE, and DB); data
acquisition, analysis, or interpretation (LT, DE, LR, NT, VB, DB,
FP, and AV); drafting the manuscript (LT, DE, LR, NT, VB, DB,
FP, and AV); and critically revising the manuscript (LT, DE, and
AV). All authors gave final approval and agreed to be accountable for
all aspects of work in ensuring that questions relating to the accuracy
or integrity of any part of the work are appropriately investigated and
resolved.
Authors’ Note
This article is a product of a Society of Toxicologic Pathology (STP)
Working Group involving the Scientific and Regulatory Policy Com-
mittee (SRPC) and Clinical Pathology Interest Group (CPIG) of the
STP in collaboration with the Regulatory Affairs Committee (RAC)
of the American Society for Veterinary Clinical Pathology (ASVCP)
and has been reviewed and approved by the SRPC and Executive
Committee of the STP.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
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