Juvenile animal testing in drug development Is it useful?

Paul Baldrick
*
Scientic and Regulatory Consulting, Covance Laboratories Ltd., England, UK
a r t i c l e i n f o
Article history:
Received 25 February 2010
Available online 27 March 2010
Keywords:
Juvenile animal
Pediatric drug development
Toxicology testing
PIP
a b s t r a c t
In pharmaceutical drug development, there has been increased interest in the need to perform juvenile
animal studies to support the safety of use of new medicines in the pediatric population. Although such
studies are not new, the increased interest has been formalized in recent regulatory guidelines. As a
result, companies are now performing many more studies in juvenile animals, even when there is a lack
of robust knowledge of cross-species functional and kinetic differences among juveniles that means
extrapolation of any toxicology study nding to an immature human may not be easy or even relevant,
especially if performed in the wrong species at the wrong time. It will be shown by presentation of some
basic considerations needed in order to perform such testing, that juvenile animal studies are indeed fea-
sible. However, it will also be highlighted that (based on available knowledge) there are currently not
enough clear-cut examples to answer the question of whether juvenile animal toxicology studies to sup-
port pediatric development (by affecting the performance or design of a pediatric clinical trial or identi-
fying a potential different-from-adult safety risk in clinical use) are truly useful or necessary.
2010 Elsevier Inc. All rights reserved.
1. Introduction
Until recently, information relating to use of medicines in chil-
dren was generally lacking leading to substantial unlicensed and
off-label use of adult drugs in the pediatric population. The situa-
tion was not helped by the fact that there was no legal requirement
for pharmaceutical companies to perform pediatric studies if there
was no intention to develop a drug for this population. However, 2
events in recent years have occurred to change thinking. Firstly,
the view that clinical trials in children are unsafe and unethical
has been superseded by realization that administration of drugs
not previously tested in children may pose a greater risk than con-
ducting studies. Secondly, there has been a shift in regulatory
thinking and specic legislation for pediatric clinical trials are
now in place in the United States (US) and Europe. Key dates in
the history of the latter legislation are given in Table 1 along with
associated regulatory guidance in Table 2. In the case of some
drugs, there is now an expectation to support the pediatric clinical
trials with juvenile animal studies. Both the guidance documents
from the USs Food and Drug Administration (FDA) and the Euro-
pean Medicines Agency (EMA) given in Table 2, which deal with
such non-clinical testing, comment that juvenile animal studies
can provide information that might not be derived from standard
adult toxicology studies or from safety information from adult hu-
mans. However, a case-by-case approach is supported and the
ICH M3 (R2) guidance relating to non-clinical safety testing (also
listed in Table 2) highlights that juvenile animal studies should
be considered only when previous animal and human safety data
are judged to be insufcient to support pediatric trials.
In interaction with either the FDA or EMA during a drugs early
development, pediatric clinical plans need to be discussed includ-
ing whether juvenile animal testing is appropriate. For the EMA,
the Pediatric Investigation Plan (PIP) as mentioned in Table 1 re-
quires description of what juvenile animal work is planned and
specic endpoints (e.g., neurotoxicity assessment), along with out-
line of the study design and timelines for the work. Experience has
indicated that even if juvenile animal work is not proposed by a
pharmaceutical company, a regulatory agency may disagree and
request such testing.
With the increased interest in juvenile animal testing to support
pediatric drug development, this paper will examine whether such
testing is truly useful. In order to achieve this goal, some difcul-
ties in the process of age-related cross-species comparison for tox-
icological endpoints will be examined, along with considerations
highlighting the need to plan any testing properly, as well as dis-
cussion of published juvenile animal study results and whether
they are of any real use in the risk assessment process for a drug
entering pediatric clinical work.
2. Adults vs juveniles
An initial consideration as to the overall utility in juvenile ani-
mal testing is knowing your animal in the period between birth
0273-2300/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.yrtph.2010.03.009
* Address: Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY North
Yorkshire, UK.
E-mail address: paul.baldrick@covance.com
Regulatory Toxicology and Pharmacology 57 (2010) 291299
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j our nal homepage: www. el sevi er . com/ l ocat e/ yr t ph
and attainment of sexual maturation, especially the fact that
experimental animals have compressed developmental schedules
compared to humans. Table 3 summaries age comparisons com-
monly used between toxicology testing species and humans. Thus,
a 2-year old child might be considered equivalent to a 21-day old
rat or a 6-week old puppy, while a rat at 47 days may be deemed
roughly equivalent to a preterm human infant. Although such com-
parison is useful as a starting point, it is not ideal (e.g., the table
does not account for organ maturation or even components within
each organ system). Useful information on major kinetic and func-
tional differences between juvenile and adult animals is, however,
gradually appearing in the literature and has been discussed else-
where (review in Baldrick, 2004; Cappon et al., 2009).
With kinetic differences between mature and immature ani-
mals, the possibility of toxic effects is introduced due to altered
metabolism (e.g., much higher drug levels due to reduced clear-
ance) compared to adults. It has been pointed out that metabolic
capability typically reaches adult levels within 612 months in hu-
mans and by weaning in rodents (Hew, 2006). An area of particular
concern in evaluation of potential CNS exposure to a drug in new-
borns is development of the bloodbrain barrier (BBB). In rats, it is
reported as immature in 1011 day old animals (Arya et al., 2006)
but established by about 2 weeks (Xu and Ling, 1994), although
other work has shown that brain-to-blood transport mechanisms
that were inoperative in the early neonatal rat become functional
in weanlings (Cornford and Cornford, 1986). In humans, the BBB
is immature in newborns and neonates becoming more effective
at 36 months (Hew, 2006).
With functional differences between mature and immature ani-
mals, the possibility of toxicity in the latter that is not seen in ma-
ture systems is introduced. Of concern is that despite the growing
knowledge for such differences between juvenile and adult ani-
mals used in toxicology studies, there are still areas in which a lack
of knowledge or understanding has the potential to compromise
assessment for true toxicity. Examples are as follows:
In terms of CNS evaluation, it has been pointed out that
although the rat and dog are appropriate models for compara-
tive CNS development, no species provides a direct correlation
to human neurological development (Wood et al., 2003). Also,
there is no precise information that equates various stages of
prepartum and postpartum brain development in humans and
animals (Brent, 2004). Older data (from the 1970s) estimated
that rat neurodevelopment at 7 days of age is equivalent to that
in a human brain at birth but others have calculated an age of
1213 days in rats (Clancy et al., 2007; Romijn et al., 1991).
Sensory and reex functions are relatively developed at birth in
humans but underdeveloped in laboratory species and adult-like
locomotion only appears around day 1516 (shortly after eye
opening) in rats, with rearing without foreleg support occurring
fromaround day 18 (Hew, 2006; Wood et al., 2003). Locomotion
in the dog develops during the rst month or so after birth, with
standing observed around 21 days of age (Wood et al., 2003).
In the kidney, nephrogenesis is complete prenatally in humans
(by gestational week 3435) but post-natally in the rat (by
11 days of age) and dog (by about 2 weeks post-natally) (Beck
et al., 2006; Hew, 2006; Zoetis and Hurtt, 2003a). Functional
development also shows species differences with glomerular
ltration rate increasing in early post-natal life in all species
as well as post-natal development of acid base equilibrium
and control of urine volume, but concentrating ability develops
prenatally in dogs (Zoetis and Hurtt, 2003a).
Table 1
Key dates in pediatric drug development.
USA (FDA, 2010)
1997: FDA Modernization Act (FDAMA) described nancial incentives for development and marketing of drugs that could be used for pediatric patients (e.g., extra
6 months of marketing exclusivity)
1998: Pediatric Rule which required pediatric studies for drugs in which signicant use or benet in children was expected
2002: Best Pharmaceuticals for Children Act (BPCA)
2003: Pediatric Research Equity Act (PREA) gave the FDA authority to require research into drugs used in juvenile patients
2007: FDA Amendments Act which re-enforced the BPCA and PREA and established the Pediatric Review Committee (PeRC)
a
Europe (EMEA, 2007)
2007: Under a EMEA initiative Better Medicines for Children a Pediatric Regulation (Nos. 1901/2006 and 1902/2006) came into force to improve the health of
children. As part of the Regulation, a PIP must be submitted and agreed by a Pediatric Committee (PDCO). The PIP facilitates the development, accessibility and
safe use of new drugs in the pediatric population through clinical studies or to provide justication for a waiver/deferral from such studies
a
a
Regulatory interaction differs between regions, a PIP needs to be submitted after initial clinical trial work, while the process of discussing pediatric studies is later in the
US when clinical efcacy has been established.
Table 2
Key regulatory guidance documents.
Global
International Conference on Harmonisation (ICH) E11: Clinical Investigation of Medicinal Products in the Pediatric Population: Covers timing of clinical studies,
study types, age classication and ethical issues (ICH, 2001)
ICH M3 (R2): Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (ICH, 2009)
USA FDA
Recommendations for Complying with the Pediatric Rule: Provides information on how to prepare a pediatric plan to comply with the FDAs Pediatric Rule (FDA,
2010)
Non-clinical Safety Evaluation of Pediatric Drug Products (FDA, 2006)
Europe
Clinical Trails Directive 2001/20/EC: Provides information for the conduct of clinical trials conducted on children (EU, 2001)
Non-clinical Testing in Juvenile Animals on Human Pharmaceuticals for Paediatric Indications (EMEA, 2008)
Table 3
Age comparisons across species.
Species Neonate Infant Child Adolescent
Rat (week) 01 13 39 9+
Dog (month) 00.75 0.751.5 1.55 5+
Primate (year) 00.05 0.050.5 0.53 3+
Man (year) 00.1 0.12 212 12+
292 P. Baldrick / Regulatory Toxicology and Pharmacology 57 (2010) 291299
For the cardiovascular system, little information is available on
cardiac output and hemodynamics in dogs although signicant
increases inbloodpressureanddecreases inheart rateoccur from
1 week to 6 months (qualitatively similar to that seen in young
infants and children) (Hew and Keller, 2003). Successful mea-
surement of electrocardiogram (ECG) and/or blood pressure has
recently been reported in juvenile dogs from as early as 4 weeks
of age (Henwood and Miller, 2004; Li et al., 2010; Perron et al.,
2009; Salvail et al., 2010; Thorsrud et al., 2010). Waveform mor-
phology was found to change with maturation (e.g., QT interval
increases with age (Li et al., 2010; Thorsrud et al., 2010) although
care in assessment is needed as high heart rate can result in dif-
culty inmeasuring routine intervals (HenwoodandMiller, 2004).
In young monkeys, successful measurement of heart rate (Day
1 + Weeks 4, 8, 13, 26 and 39) and blood pressure (Week 13 and
39) has been reported (Rasmussen et al., 2007).
For the respiratory system, rat lungs reach alveolar stage at
4 days of age then develop rapidly with most development
complete within 24 weeks after birth while humans are born
with lungs in the alveolar stage and development continues to
age 28 (Beck et al., 2006; Viau and Robinson, 2006; Zoetis
and Hurtt, 2003b). Conicting data occur for dogs with reports
that the lung is either slightly less mature at birth than human
lung (e.g., the degree of lung maturity at day 11 in dogs to be
comparable to humans at birth) or more mature at birth; alve-
olar development in the dog is complete by 16 weeks of age
(Viau and Robinson, 2006; Zoetis and Hurtt, 2003b). In the pri-
mate, lung development is advanced at birth compared to
humans with completion from one year of age (Beck et al.,
2006; Hew, 2006; Zoetis and Hurtt, 2003b).
In order to compare the gastrointestinal tract, it is important to
know that it is more mature in humans at birth than in the rat,
with maturation in the rodent occurring during the rst
3 weeks after birth (Walthall et al., 2005).
Overall, the lack of robust knowledge of the systems described
above in juvenile animals means that extrapolation of any toxicol-
ogy study nding to an immature human may not be easy or even
relevant especially if performed in the wrong species at the wrong
time. Thus, when examination of a toxicity of potential concern is
thought necessary in a juvenile animal, its age at dosing needs to
be considered to allowtreatment at the appropriate developmental
stage in time with maturation of the target organ of concern. Dif-
ferent dose levels compared to adult animals may also be needed
if exposure is shown to be higher or lower. To investigate potential
human neonate pulmonary effects, it has been shown recently that
nose-only inhalation dosing of rats aged 421 days of age is feasi-
ble, a period depicting human lung development stages between
preterm newborns and children of about 68 years of age (Lewin
et al., 2008). For pre-weaning animals, there is a need to consider
developmental landmarks e.g., in rats, physical development (pin-
na detachment, eye opening, tooth eruption, vaginal opening, pre-
putial separation) and sensory and reex development (negative
geotaxis, righting reex, visual placing, acoustic startle, papillary
reex). Experience shows that an apparent effect on physical or
functional development may solely be due to a drug-induced effect
on bodyweight. The lack of locomotion in very young rats means
that a full panel of functional observational battery (FOB) parame-
ters (e.g., gait and rearing) used for CNS evaluation cannot be as-
sessed and thus allow comparison to adult data.
A further concern involves potential regulatory requests for
assessment of immunotoxicity in juvenile animals, even if no par-
ticular drug-induced adverse immune system signal was seen in
adults. It has been pointed out that for humans, information on
expression of immune endpoints in juveniles is lacking and it is
not known whether juvenile humans are more sensitive to im-
mune system perturbations than adults (Holsapple et al., 2005).
Potential immunotoxicity can indeed be measured in toxicology
studies (as for adult animals) from the following endpoints: (1)
standard white blood cell count, spleen and thymus weights + his-
topathology and (2) response to T lymphocyte-dependent antigen
challenge and/or lymphocyte subpopulation examination in spleen
cells. Many of these measurements are reported as possible in rats
from weaning (Barrow and Ravel, 2005). However, actual data on
the rat immune system are limited, although it is known to be
developmentally delayed relative to humans, with considerable
anatomical development occurring after birth (e.g., a reduced im-
mune response occurs at approximately 3 weeks vs adults) (Hols-
apple et al., 2003). Histologically, it has been shown that there is
little indication of immunologic stimulation in the immune system
of rats at 3 weeks (Parker et al., 2008). Limited data are also avail-
able on the status of the immune system of juvenile non-rodents,
although it is reported that it is fully developed before birth (dogs)
or, as in humans, well developed at birth (macaques) with maturity
in both species continuing into post-natal life (Martin and Buse,
2008; Felsburg, 2002). However, full information on comparable
times of functional immuno-competence is scant. These species
differences again raise the question of when to assess for immuno-
toxicity (and indeed, if needed) in juvenile animals and how to
interpret ndings.
3. Study considerations
As reported elsewhere, toxicology studies in very young ani-
mals are not new and they have been performed for certain drug
classes in the past to assist pediatric development (Baldrick,
2004). Indeed, a compilation of lethal dose (LD)
50
values in new-
born and adult animals was produced in the early 1970s (Golden-
thal, 1971). However, more detailed information is becoming
available on study considerations associated with performing work
in juvenile animals (Bailey and Marien, 2009; Baldrick, 2004; Bar-
row, 2007; Beck et al., 2006; Hew, 2006; ILSI, 2003; Moser et al.,
2005) and examples of possible study designs have recently been
published (Cappon et al., 2009). Considerations include:
Species of use: The rat is the usual species of choice (see later).
As a result of performing reproduction toxicology studies, labo-
ratories have experience of working with newborn and young
rats. Much less experience is available for juvenile studies in
dogs, primates and minipigs (in fact, although some companies
can perform these studies with puppies in-house, only a small
number of Contract Research Organizations [CROs] offer testing
in all 3 species). It has been pointed out that small sample size,
high inter-animal variability, limited historical data and a high
risk of maternal rejection in pre-weaned animals makes inter-
pretation of results from toxicology studies using juvenile pri-
mates very difcult (Hew, 2006). However, studies in non-
rodents have been performed when target organ toxicity is
not seen in the rat or, when the rodent it is not a pharmacolog-
ically relevant species.
Age of animals at start of treatment: Oral dosing of animals can
commence at one day of age, although experience has indicated
increased levels of mortality among rats (with a weight of
approximately 56 g) at this age (e.g., from dosing error even
when using a dose volume of 10 mL/kg which would result is
administration of 0.05 mL). Indeed, mortality can also occur at
4 days of age and in the authors laboratory it is recommended
that this is the earliest age to start oral dosing in rats, unless
there is a strong reason for starting earlier. Dosing of pre-
weaned rats is technically more complex than in weaned
animals (including the question of dosing vehicle and different
P. Baldrick / Regulatory Toxicology and Pharmacology 57 (2010) 291299 293
levels of drug to whole, split or cross-fostered litters and result-
ing consideration for cross-group drug contamination). In the
authors laboratory, it was found that initial dosing of 3-week
old rat pups (to cover the human age of 2 years see Table 3)
is better performed on Day 22 and not at weaning on Day 21
to reduce potential stress. Concurrence on an optimal age for
juvenile toxicology studies in primates is not available but an
age range between 9 and 36 months is reported as generally
appropriate and practical (Chellman et al., 2009).
Numbers of animals: A sufcient number of pregnant animals
are needed to produce enough offspring for both the range-nd-
ing and main toxicology studies. Depending on the age at start
of dosing, rats can be supplied as dams with litters (e.g., culled
to 4 males and 4 females per litter) from the breeder. An addi-
tional consideration with the use of non-rodents is the difculty
in getting enough offspring available at one time (necessitating
staggered dosing regimens and long study durations) and the
issue of what to do with the breeding stock. The challenge of
procuring adequate numbers of appropriately aged monkeys
of suitable gender has been mentioned elsewhere (Chellman
et al., 2009). These authors also comment that juvenile cyno-
molgus monkeys can be acquired from vendors at approxi-
mately 89 months (post-weaning), younger animals may be
available but would need to be shipped with their mothers.
Toxicokinetic evaluation: It is important to demonstrate how
similar or different exposure is between juveniles and adults
but due to the small blood volume in rats, terminal sampling
(e.g., from the aorta) is needed for very young animals. It is
practical, if possible, to keep evaluations to a minimum at this
early age (e.g., to give an estimate of maximum concentration
and area under the curve), otherwise a large number of satel-
lite animals will need to be included in the study purely for
toxicokinetic assessment. In a recent example in the authors
laboratory, groups of 18 male and 18 female rats were needed
for an early evaluation, as only one terminal sample can be
taken (up to about 3 weeks of age) followed by groups of 9
males and 9 females for older animal evaluation (2 bleed times
per rat), as in routine toxicology studies with adult rats (Bald-
rick, 2008).
Study duration and dose levels: As toxicology data will be
available in young adult animals (with dosing commencing
from 610 weeks of age in rats and 610 months in age in
dogs), treatment is usually only needed to reach this time
point (or the age at sexual maturity). A common dosing period
in the rat is from around Day 21 to up to 70 of age, sometimes
with a further non-dose recovery period and/or assessment of
reproduction performance. Range-nding work is usual to set
the high dose level for the main toxicology study but it is
important to avoid marked toxicity which will compromise
interpretation of ndings (e.g., growth retardation with sec-
ondary effects on organ systems and/or maternal neglect/
rejection of offspring).
Background data: Although a concurrent vehicle control group
will be used in each juvenile animal toxicology study, there is
sometimes a need to refer to historical control data to aid inter-
pretation of a nding. Thus, if a study is to be performed at a
CRO, then it is important to ensure that it has enough experi-
ence with juvenile animal testing to have such data (e.g., long
bone growth values) if needed. In recent years, clinical pathol-
ogy data have been published for juvenile rats and dogs (Beck
et al., 2006; Hew, 2006; Pinsonneault et al., 2008). This data
shows notable age-related changes occur in hematology and
clinical chemistry parameters. Interestingly, in a recent survey
of standard clinical pathology evaluation in 31 juvenile animal
toxicology studies, no increased sensitivity was seen in the
immature vs the adult animal (Bailey and Marien, 2009). Based
on this dataset, it was concluded that clinical pathology evalu-
ation is of little value and should only be considered if it is a tar-
get for the drug (and not included as standard).
Is there a specic toxicity that needs to be evaluated and, as
pointed out earlier, is enough known about the organ system
involved to include in the study and properly assess?
4. Juvenile animal study frequency
As a result of the changes in regulatory thinking, juvenile ani-
mal studies are increasingly in demand. An evaluation of EMA
PIP applications showed that 340 decisions were published (to
23 December 2009) for about 266 drugs (some applications were
for the same drug) (EMA, 2010). Furthermore, the outcome of some
recent PIP submissions has not yet been published due to ongoing
discussions between the EMA and the applicant company to seek
agreement on the nature of requested work. A total of 45 (17% of
drugs) of the published decisions had juvenile animal work re-
quested (to support the pediatric age range of from birth-12 years
to less than 18 years). The therapeutic areas involved are shown in
Table 4, with the most popular areas being oncology, cardiovascu-
lar diseases and infectious diseases. Unfortunately, due to the con-
dential nature of the data, the rationale behind the requests for
these studies is not in the public domain (e.g., it would be interest-
ing to see why oncology products are of particular concern to the
regulators to result in the joint most frequently requested number
of studies to support pediatric development). It is also unknown
how many of the requested studies were actually made by the
applicant companies themselves based on safety concerns for pedi-
atrics vs a direct request from the EMA regulators.
The information showed that 29 (64% of affected drugs) deci-
sions specied rat, 4 specied dog, 3 specied monkey, 2 specied
mouse, 1 specied sheep + 8 had species not specied. Only 3
drugs had a request for testing in 2 juvenile species. Most of the
drugs were small molecules although some biological drugs were
included (4 monoclonal antibodies and 2 recombinant proteins).
The requested studies in dogs were for indications of epilepsy (2
drugs), asthma and parathyroid carcinoma/hyperthyroidism, while
the studies in monkeys were for fungal infections, psoriasis (a
monoclonal antibody drug) and blood clotting deciency (note this
latter monkey study appeared to be a pharmacokinetic, not toxicol-
ogy, assessment in juvenile monkeys). Published information on
juvenile animal requests involving the US FDA are generally lack-
ing. However, it has been stated that more than 110 juvenile ani-
mal studies have been proposed/conducted in the past 8 years in
a drugs Investigational New Drug (IND) stage (Jacobs, 2009).
Table 4
Therapeutic area for which juvenile animal studies have been requested in Europe
based on PIP submissions.
a
.
Therapeutic area Number of animal studies (out of total of 45 drugs)
Oncology 7
Cardiovascular
diseases
7
Infectious diseases 6
Endocrinology 5
Pneumology 5
Immunology 4
Neurology 3
Psychiatry 2
Dermatology 1
Gastroenterology 1
Hematology 1
Ophthalmoscopy 1
Pain 1
Uro-nephrology 1
a
Up to December 2009 (EMA, 2010).
294 P. Baldrick / Regulatory Toxicology and Pharmacology 57 (2010) 291299
5. The utility of juvenile animal studies
Do juvenile animal studies actually identify any new toxicities
that would affect the performance or design of a pediatric clinical
trial or identify a potential different-from-adult safety risk in clin-
ical use? In a recent (albeit limited) survey, data from 39 juvenile
toxicology studies conducted by 10 pharmaceutical companies
was published (Bailey and Marien, 2009). A total of 29 studies were
performed in rat, with 9 studies in dog and 1 study in minipig. In
assessing sensitivity (i.e., a treatment effect), it was found that
overall the juvenile animal showed a tendency towards being more
sensitive (unfortunately the publication did not speculate on
whether or not this nding was possibly due to higher systemic
exposure/altered metabolic capacity). The study authors concluded
that, in general, ndings in the majority of these studies could have
been predicted from either the known pharmacology or adult data.
Novel toxicity was seen in 4/39 studies (one CNS and 3 anti-
infective drugs). Although none of the novel ndings were predict-
able from adult data, they were found from histopathological
examination (and animal observations in one case) and not as a
result of any sophisticated or complex design. Unfortunately,
the publication does not expand on these toxicities (due to the con-
dential nature of the data) but it may be that they were purely
due to maturation and/or kinetic differences and not novel toxici-
ties at all. Recent examples of maturation differences are available
in the literature. Table 5 shows an example of testicular ndings in
a juvenile rat toxicology study with a non-steroidal anti-inamma-
tory drug (NSAID), which were not seen in adults; they were re-
lated to developmental differences (Anderson et al., 2009).
Cardiotoxicity (arteriopathy) in rats dosed with cyclosporine at a
young age (428 days of age), but not in healthy adult animals,
has been related to a possible unique vulnerability of juvenile rats
(Allais et al., 2009). The recent literature also has examples of tox-
icity in juveniles that was not seen in adults due to much higher
exposure. Thus, toxicity (in the form of death) seen in juvenile rats
(dosed on days 1225 of age) but not in older animals, was related
to a 3-fold higher exposure (Bailey and Marien, 2009). Study nd-
ings in other juvenile animal work with the drug were no different
to that seen in the adult at comparable systemic exposure. In an-
other example with an anti-infective drug in early clinical develop-
ment, it was shown that single oral doses to rats of 1 and 15 days of
age resulted in 15 and 7-fold higher exposure, respectively, than in
adults, while oral bioavailability was close to 100% at day 15 vs
only 24% in adults (De Schaepdrijver et al., 2008). Repeated oral
administration of the drug also showed 35-fold higher exposure
at day 18 vs day 24 and it was felt that the differences may have
been related to a low capacity of metabolizing enzymes in younger
animals, with reduced rst pass metabolism.
There is still a limited database of published recent studies in
juvenile animals. However as a snap-shot, Table 5 summarizes
juvenile animal toxicology study data for 12 drugs (including that
available on the product label of 7 of them) and examines
whether the studies were actually useful in identifying a toxicity
that would have affected the human (pediatric) risk assessment
process. It is not clear if the answer is yes. As pointed out in
the table, real juvenile animal ndings were seen for CNS active
drugs such as Prozac, Strattera, Vyvanse and Ritulin, but it is un-
clear what they actually mean for pediatric use of the drug, other
than the recommendations made in the product label. In fact, it
may be that animal neurobehavioral ndings would have been
predicted based on the pharmacology of the drugs? It is also
not clear why dosing of juvenile rats started at such an early
age (621 days of age) for these drugs, unless the lack of informa-
tion on comparative CNS development between rodents and hu-
mans mentioned earlier was a consideration. Interestingly, in an
recent example of a study design for a drug to treat attention def-
icit hyperactivity disorder (ADHD), dosing in rats was indicated
from day 7 of age, with this early start stated to support the
youngest age for the intended pediatric population (Cappon
et al., 2009) and considered appropriate for testing of potential
safety risks in children of age 612 (Anderson et al., 2009). In a
further case for another CNS active drug intended for chronic
treatment occurring in newborns or premature births, dosing in
dogs from day 4 of age was indicated (Cappon et al., 2009). In a
recent survey, positive ndings were only observed in 2 juvenile
animal studies with CNS drugs out of 26 studies in which behav-
ioral testing was conducted (Bailey and Marien, 2009). The study
authors question whether the positive ndings were actually
showing subtle changes in the animals behavior. For other drugs
such as Dolobid, the signicance of juvenile animal ndings is un-
clear in the risk assessment process. Interestingly, the same pat-
tern of neonatal toxicity (including death, an effect on
bodyweight and cataracts) was reported in rats and puppies with
the widely used drug aspirin, which was used as a comparator in
this work. The current product label for aspirin states Consult
your doctor before treating a child younger than 12, a require-
ment not related to juvenile animal work but to the rare occur-
rence of Reyes syndrome in this population (Rxlist-1, 2010). It
is not known if the ndings with these NSAIDs were purely re-
lated to much higher exposure in the juvenile animal? In the case
of Tamiu, the enhanced neonatal rat toxicology was related to
greatly increased brain (and plasma) exposure, which could be
questioned as whether a novel toxicity or not.
Close examination of the FDAs guidance on non-clinical testing
(see Table 2) also indicates that the regulators (at least in 2006
when the document was nalized) may not be totally convinced
of the utility of juvenile animal work. The guidance gives some
examples where studies in young animals may be useful in the
prediction of age-related toxicity in children or showed drug-in-
duced post-natal developmental toxicity. Examples in the rst
category include phenobarbital effects on the developing rodent
nervous system (= association with altered cognitive performance
in children?) or theophylline pro-convulsant effects in developing
rodents (= association with an increased risk of convulsion in
young children?). Examples in the second category comprised
chondrotoxicity in immature animals treated with uoroquino-
lones, long-term changes in serotonergic innervation in juvenile
rats exposed to uoxetine or effects of methylphenidate on growth
and endocrine function in young rats. However, for all the exam-
ples given, it is stated that the signicance of these ndings for
humans is uncertain. It might be argued that a well-designed
juvenile animal toxicology study would have been useful to sup-
port pediatric development and helped to prevent adverse ndings
in children with some of these drugs. However, based on the re-
sults of such studies with 2 of the drugs (uoxetine and methyl-
phenidate) as given in Table 5, it is unclear if they were
additionally helpful in the risk assessment process.
As indicated in Table 5, the key question is what does a positive
nding in a juvenile animal study mean? A few examples are:
Effects in CNS testing in juvenile rats (e.g., a decrease in open
eld motor activity and decit in a passive avoidance test) at
small margins of clinical dose real potential for neurological
effect in children?
Effect on long bone growth in juvenile animals (not likely to be
seen in adults as growth is complete) concern for children?
Delayed/altered sexual maturation in rats (e.g., effect on prepu-
tial separation, vaginal opening and reduced sperm count/mor-
phological changes) (not likely to be seen in adults as they are
sexually mature) concern for children?
P. Baldrick / Regulatory Toxicology and Pharmacology 57 (2010) 291299 295
Table 5
Examples of juvenile animal toxicology studies with a question on their usefulness.
Drug (therapy) Study design/ndings Outcome from study Question on study Reference
Marketed NSAID Tested in juvenile rats (PND 752)
and dogs (PNW 1030)
Non-dose related efferent occlu-
sions and seminiferous tubule
dilatation + increased testis weight
in a few rats but no effect on
spermatogenesis
Not seen in juvenile dogs + repro-
duction work in adult rats at toxic
doses showed no effect on male
fertility, pathological changes or
effect on testis weight
Review of anatomy and physiology of
efferent ducts and developmental
differences vs other species including
man concluded that the nding was
specic to juvenile rats and not
relevant to human assessment of
reproductive risk
What did these studies add to the risk
assessment process?
Anderson
et al. (2009)
Levetiracetam (anti-
epilepsy drug)
Assessment in juvenile rats at up
30-fold the maximum recom-
mended human oral dose
No behavioral effects found
Concluded that this favorable result
may be one of many key factors to
consider when choosing an epilepsy
treatment in pediatrics
Did this work actually add to the risk
assessment process?
Leonard et al.
(2008)
Dolobid (diunisal NSAID
for treatment of pain)
At dose levels
ca
3-fold the usual
human therapeutic dose, both
Dolobid and aspirin resulted in
death, leukocytosis, bodyweight
loss and cataracts in neonatal pup-
pies (45 day old) after 210 doses
while lower doses to 25 day old
puppies resulted in lower mortal-
ity, and did not produce cataracts
In newborn rats, Dolobid adminis-
tration resulted in decreased
bodyweight gain while aspirin
resulted in increased mortality
and some cataracts
Product label states that safety and
effectiveness in pediatric patients
below the age of 12 have not been
established + use in pediatric patients
below the age of 12 is not
recommended
Did the juvenile animal work add to
the risk assessment process?
Rxlist-2
(2010)
Strattera (atomoxetine
treatment of ADHD)
Growth, neurobehavioral and sex-
ual development evaluated in
young rats at up to
ca
8-fold the
maximum human dose from PND
10 through to adulthood
Slight effects were seen (delays in
onset of vaginal patency and pre-
putial separation, decreases in epi-
didymal weight and sperm
number, decreases in corpora
lutea, delay in onset of incisor
eruption and increased motor
activity)
However, there were no effects on
fertility or reproductive perfor-
mance learning and memory tests
Product label states that the
signicance of these ndings to
humans is unknown + safety, efcacy
and pharmacokinetics in pediatric
patients less than 6 years have not
been established
Did the juvenile rat work add to the
risk assessment process?
Rxlist-3
(2010)
Vyvanse (lisdexamfetamine
treatment of ADHD)
Juvenile rats received oral doses
on PND 763 at approximately
0.3, 0.7 and 3 times the maximum
recommended human daily dose.
Dose-related decreases in food
consumption, bodyweight gain
and crown-rump length were
seen; after a 4 week drug-free
recovery period, bodyweights and
crown-rump lengths had signi-
cantly recovered in females but
were still substantially reduced in
males
Time to vaginal opening was
delayed in females at the highest
dose, but there were no drug
effects on fertility when the ani-
mals were mated beginning on
day 85 of age
Juvenile dogs were dosed for
6 months beginning at PNW 10
and showed decreased body-
weight gain at approximately 0.5,
1 and 3 times the maximum rec-
ommended human daily dose. This
effect partially or fully reversed
during a 4 week drug-free recov-
ery period
Product label states that the drug is
indicated for use in children aged 6 to
12 years. It has not been studied in
children under 6 years of age or
adolescents
Did the juvenile animal work add to
the risk assessment process?
Rxlist-4
(2010)
296 P. Baldrick / Regulatory Toxicology and Pharmacology 57 (2010) 291299
Table 5 (continued)
Drug (therapy) Study design/ndings Outcome from study Question on study Reference
Ritalin (methylphenidate
treatment of ADHD)
Oral administration to rats from
PND 7) until sexual maturity
(PNW 10)
When these animals were tested
as adults (PNW 1314), decreased
spontaneous locomotor activity
was observed in both sexes at a
previously treated of from approx-
imately 6-fold the maximum rec-
ommended human dose (MRHD)
A decit in the acquisition of a
specic learning task was also
seen in females previous exposed
to 12-fold the MRHD
The no effect level for juvenile
neurobehavioral development in
rats was given as 5 mg/kg/day
(which was half the MRHD
More details of this work are also
available in the literature (Beck-
man et al., 2008)
The product label states that the drug
should not be used in children under
6 years of age. It also states that the
clinical signicance of the long-term
behavioral effects observed in rats is
unknown
Did the juvenile rat work add to the
risk assessment process?
Rxlist-5
(2010)
Mevacor (lovastatin used
for cholesterol lowering)
Dosing of neonatal rats by subcu-
taneous injection resulted in
delayed passive avoidance learn-
ing in females but not males at
exposures up to 4-fold human sys-
temic exposure
(Note: subcutaneous route used to
increase exposure by avoiding
stomach lesions seen in other tox-
icology studies by oral route)
Product label states the drug has not
been studies in pre-pubertial patients
or patients less than 10 year of age
Did the juvenile rat work add to the
risk assessment process?
Rxlist-6
(2010)
Bridion (sugammadex)
neuromuscular blocking
agent used in
anaesthesia)
Binds to bone and teeth but no
adverse effects seen in general
toxicology studies
However, testing in juvenile rats
resulted in incisor discoloration
and disturbances of amelogensis
It was concluded that the large safety
margin seen for this effect indicated
that the risk of teeth/bone growth
and development effects occurring in
the pediatric population was low
(especially given the conditions of
clinical use single dose + in
conjunction with an aminosteroidal
drug)
Did the juvenile animal work add to
the risk assessment process?
Bridion EPAR
(2008)
Prozac (uoxetine major
depressive
disorder + obsessive
compulsive disorder in
adults and pediatric
patients from 7 years)
Signicant toxicity including myo-
toxicity, long-term neurobehav-
ioral and reproductive toxicity
and impaired bone development
were seen in juvenile rats dosed
from PND21 to adulthood (PND
90)
These adverse ndings were not
seen in adult rats
Impaired bone formation and
abnormal emotional behaviors
also seen in juvenile mice
Some of the effects occurred at
clinically relevant exposures
The product label states that anyone
considering the use of Prozac in a
child or adolescent must balance the
potential risks with the clinical need.
Short-term clinical studies showed
no differences in ndings between
adults vs pediatrics
Obviously real juvenile animal
ndings but what did they actually
mean for pediatric use of the drug?
Rxlist-7
(2010)
Tamiu (oseltamivir
treatment of inuenza
infection)
A single dose of 1000 mg/kg to 7-
day old rats resulted in deaths;
no deaths were seen at 2000 mg/
kg in 14-day-old rats
It was found that drug concentra-
tion in the brain was ca
1500-fold
higher in 7-day old vs adult rats
administered 1000 mg/kg (levels
in plasma were 10-fold higher
compared with adults). Results
suggested that brains levels of
drug decrease with increasing age
and most likely reect the matura-
tion stage of the BBB
No adverse effects occurred at
500 mg/kg/day administered to 7-
to 21-day-old rats. At this dosage,
exposure was
ca
800-fold that
expected in a 1-year-old child
The product label states the drug is
not indicated for pediatric patients
younger than 1 year of age
Obviously important nding for
human infant safety but due to
kinetics not toxicology, so any use in
juvenile toxicology work?
Rxlist-8
(2010)
(continued on next page)
P. Baldrick / Regulatory Toxicology and Pharmacology 57 (2010) 291299 297
6. Conclusion
We know that toxicology studies can be conducted in very
young animals, although there is still a limited database of informa-
tion in this age group. However, a key question is, do they investi-
gate ndings that cannot be adequately and safely assessed in
pediatric clinical trials, examine toxicity not covered in adult stud-
ies (toxicology as well as pharmacology and kinetic evaluation),
investigate added parameters for safety evaluation in pediatric
work or help with drug product labeling? Even if the answer is lar-
gely no, it could be argued that a lack of new ndings in a juvenile
animal toxicology study is a positive result in that it conrms a lack
of increased sensitivity for young children to the drug being tested.
Other authors have also questioned the need for inappropriate or
unnecessary studies being performed or the inclusion of parame-
ters, which generate little or no useful information (De Schaepdrij-
ver et al., 2008) or the performance of complex, inappropriate or
unnecessary studies or in the inclusion of complex parameters that
generate little or no newuseful data (Bailey and Marien, 2009). The
case-by-case approach for juvenile animal studies has been en-
dorsed in a recent publication (Cappon et al., 2009).
While it is true that the results of the recent limited assessment
of 39 juvenile animal studies mentioned earlier reported novel tox-
icity in 4 cases, it is currently not clear if there are many (or genu-
inely any?) clear examples when juvenile animal toxicology
studies have accurately predicted novel human toxicities that
would have an impact in pediatric medicine. However, in one area
at least (CNS active drugs that have prolonged use in a pediatric
population), experience has shown that regulators still seem con-
vinced that neurobehavioral assessment in animals treated from
an early age has some utility in assessing safety in young humans.
There is also the issue of how to actually interpret study ndings
(especially if different-from-adult data) as it may be that the
majority of different toxicities between adults vs juveniles are
related purely to immaturity (e.g., through different kinetics). In-
deed, this could have been the case for the 4 previously mentioned
novel toxicities. From a kinetic perspective, if there is a real con-
cern for a safe starting dose in a pediatric clinical study (that can-
not be answered from extrapolation from adult data), there may be
a need to examine for potential exposure differences in limited
pharmacokinetic evaluation in a juvenile animal or perhaps from
toxicokinetic assessment in a toxicology study with a reduced
study design.
Despite these reservations, regulators are continuing to request
juvenile animal studies. Indeed, as mentioned earlier they have
deemed that at least 45 of these studies (although it is not known
how many were suggested by the regulators vs applicant compa-
nies themselves) for a range of drug classes (including 4 biological
drugs) were necessary to support pediatric drug development in
Europe over the last few years. Even if all these studies are indeed
needed due to safety concerns, the reported drift from a case-by-
case approach to a standard design one within both regulatory
agencies and industry (Bailey and Marien, 2009; Cappon et al.,
2009) is worrying. The challenge is convincing fellow scientists/
regulators that toxicology studies in juvenile animals should only
be performed if strictly needed and push-back to regulators for
requested work (if not felt fully justied) needs to occur. Animal
use (especially in puppies or young monkeys) with no clear goal
for risk assessment is totally unacceptable.
Current guidelines indicate a need for juvenile animal studies
only when previous animal and human safety data are judged
insufcient. This paper concludes that (based on available knowl-
edge) there are currently not enough clear-cut examples to answer
the question of whether juvenile animal toxicology studies to sup-
port pediatric development are truly useful or necessary. Maybe as
more data become available and are published, this situation will
change.
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