Juvenile Toxicology: Relevance and Challenges for Toxicologists and Pathologists

Abstract

The Society of Toxicologic Pathology (STP) Education Committee and the STP Reproductive Special Interest Group held a North Carolina regional meeting entitled, “Juvenile Toxicology: Relevance and Challenges for Toxicologists and Pathologists” on March 13, 2015, at the National Institute of Environmental Health Sciences/National Toxicology Program in Research Triangle Park, North Carolina. The purpose of this regional meeting was to familiarize attendees with the topic of juvenile toxicity testing and discuss its relevance to clinical pediatric medicine, regulatory perspectives, challenges of appropriate study design confronted by toxicologists, and challenges of histopathologic examination and interpretation of juvenile tissues faced by pathologists. The 1-day meeting was a success with over 60 attendees representing industry, government, research organizations, and academia.

Juvenile toxicity testing has become a hot topic in recent years with the institution of the Best Pharmaceuticals for Children Act (BCPA) and the Pediatric Research Equity Act (PREA) in 2002 and 2003, respectively. These acts resulted in the need for pharmaceutical safety assessment in pediatric or juvenile populations (U.S. Congress 2001; U.S. Congress 2003). The purpose of this regional meeting was to familiarize attendees with the topic of juvenile toxicity testing and discuss relevance to clinical pediatric medicine, regulatory perspectives, challenges of appropriate study design, and histopathologic interpretation of juvenile tissues. The meeting provided a public forum in which scientists from various fields could discuss strategies for addressing the many complexities of juvenile toxicology. The meeting organizing committee was composed of Thomas J. Steinbach, DVM, DACVP, DABT, director of the North Carolina Laboratory of Experimental Pathology Laboratories, Inc.; Darlene Dixon, DVM, PhD, DACVP, group leader of the Molecular Pathogenesis Group of the National Toxicology Program Laboratory (NTPL) and National Institute of Environmental Health Sciences (NIEHS); and Amera K. Remick, DVM, DACVP, DABT, assistant director of pathology at WIL Research. The organizing committee divided the meeting into 2 sessions: (1) clinical, regulatory, and toxicology perspectives and (2) pathology perspectives; and identified and invited 8 skilled professional speakers to address these diverse aspects of juvenile toxicology. A brief postmeeting synopsis was provided in the National Toxicology Program (NTP) Update Newsletter (Catlin and Quist 2015).

Opening Remarks and Clinical, Regulatory, and Toxicology Perspectives

Opening remarks for the meeting were provided by John R. Bucher, PhD, associate director of the NTP and director of the NTP Division. Dr. Bucher discussed the NTP’s activities surrounding assessment of juvenile toxicity. A conceptual shift has occurred in the last decade in which the NTP has begun to study hormonally active compounds that exhibit juvenile effects derived from gestational exposures. The NTP has also begun to replace reproductive assessment by continuous breeding (RACB) studies with modified one-generation (MOG) studies, which capture the early life exposure period through perinatal dosing (Foster 2014). The MOG studies provide an efficient design that consolidates multiple studies into 1 study and assigns pups to several testing cohorts in order to maximize information gained. This paradigm shift at the NTP has inspired new testing strategies that encourage pathologists to examine early life exposures and interpret new data sets from juvenile animals.

The clinical, regulatory, and toxicology perspectives session commenced with a brief introduction by Dr. Steinbach and a lively presentation on the clinical relevance of juvenile toxicity studies by David B. Peden, MD, MS, University of North Carolina School of Medicine, director of the Center for Environmental Medicine, Asthma and Lung Biology; chief of the Division of Pediatric Allergy, Immunology, and Rheumatology; and Andrews Distinguished professor of Pediatrics, Medicine and Microbiology/Immunology and Toxicology. Dr. Peden discussed the challenges faced by pediatricians in extrapolating drug safety information to children. This extrapolation occurs when drugs are approved for use in adults and used off-label in children. Even when preclinical juvenile animal studies are performed, clinicians must frequently make assumptions based on data collected from different or inappropriate life stages where the true toxic potential of a compound might be missed (Soellner and Olejniczak 2013). These pediatric drug safety concerns are not limited to pharmaceuticals, but can also be critical when food or environmental toxicants are “unintended drugs” in developing children (Donohue et al. 2013; Du Toit et al. 2015; Gascon et al. 2015; Midoro-Horiuti et al. 2010; Miller and Peden 2014; Petzold et al. 2014; Strobel and Ferguson 1984). Another consideration for pediatric safety assessment and study design is the increasing use of long-term pharmaceutical interventions, where continuous exposure may result in differential toxicities over the lifetime of the individual (Belvisi et al. 2005; Pedersen et al. 2010; Pruteanu et al. 2014; Zhang, Prietsch, and Duchame 2014).

From a historical perspective, as highlighted by both Drs. Peden and Elayan, pharmaceutical treatment in children based on extrapolation from adult data has been the standard, and concerns regarding the ethics or feasibility of clinical trials in children have hindered forward movement in pediatric safety assessment. Unfortunately, many drug safety incidents negatively impacting children could have been prevented with appropriate regulatory measures and testing. Ikram Elayan, PhD, is a senior pharmacology/toxicology reviewer for the Division of Psychiatry Products at the Food and Drug Administration (FDA), Center for Drug Evaluation and Research (CDER). Dr. Elayan presented a background on pediatric drug regulation and a regulatory perspective on the importance of performing well-designed juvenile animal toxicity studies with appropriate endpoints. In recent years, as regulatory agencies have begun to encourage pediatric safety assessments for pharmaceuticals, a shift has occurred from voluntary testing with the BPCA in 2002, which offered an additional 6 months of marketing exclusivity in exchange for performing pediatric safety studies, to mandatory testing with the PREA in 2003 (U.S. Congress 2001; U.S. Congress 2003). Both acts were reauthorized with the FDA Amendments Act (FDAAA) in 2007 and made permanent with the FDA Safety and Innovation Act (FDASIA) in 2012. Thus, sponsors must submit a pediatric study plan within 60 days of the end-of-phase 2 FDA meeting for any pharmaceutical intended for use in humans ≤12 years of age (U.S. Congress 2007; U.S. Congress 2012). Nonclinical juvenile animal studies are a vital component of the pediatric study plan and are used to support data for the age-group between weaning and adulthood. These studies target pediatric safety concerns that are not adequately addressed in general toxicity studies and cannot be adequately or ethically studied in all instances in pediatric clinical trials (Tassinari et al. 2011). Nonclinical juvenile animal toxicity studies should be scientifically justified and well designed with appropriate endpoints (Bolon et al. 2013; R. M. Parker 2014b; Rao et al. 2011).

The intricacies and challenges of creating well-designed nonclinical juvenile toxicity studies were addressed by Robert M. Parker, PhD, DABT, director of Developmental and Reproductive Toxicology at Huntingdon Life Sciences. Dr. Robert Parker’s seemingly simple statements of “Children are not small adults. Rats are not humans. Each pediatric age group is different” quickly developed into a detailed discussion of the logistical complexities of juvenile animal studies (R. M. Parker 2014a, 2014b). Juvenile toxicity studies involve a large number of animals, numerous and diverse study endpoints, and often multiple cohorts of animals in order to adequately address these endpoints. Factors such as latency of effects, litter effect (best controlled with a cross-fostering method), dosing of pups as young as postnatal day (PND) 1, pooled blood collections, dosing adjustments based on daily body weights due to rapid growth rate, and pharmacokinetic and pharmacodynamic differences in young animals must all be considered and handled with appropriate scientific and technical expertise and resources. With the many factors involved, it is difficult to have a standardized juvenile toxicity study design; thus, each study must be designed and performed on a case-by-case basis. Multiple references were provided for additional information on comparative organ system development (Beckman and Feuston 2003; Hew and Keller 2003; Holsapple, West, and Landreth 2003; Marty et al. 2003; Walthall et al. 2005; Watson et al. 2006; Wood, Beyer, and Cappon 2003; Zoetis and Hurtt 2003a, 2003b; Zoetis et al. 2003), developmental and reproductive toxicology (Bailey et al. 2009; Greaves 2012; Haschek, Rousseaux, and Wallig 2013; Hoberman and Lewis 2012; Hood and Parker 2008; Lerman et al. 2009; R. M. Parker 2014a, 2012; R. M. Parker and York 2013; Wise et al. 2009; York et al. 2014), and juvenile toxicology (Bailey and Mariën 2011; Cappon 2011; Cappon et al. 2009; Carleer and Karres 2011; Hurtt 2011; Leconte et al. 2011; R. M. Parker 2014b; Rose 2011; Shimomura 2011; Tassinari et al. 2011).

LaRonda L. Morford, PhD, director of Juvenile Toxicology at WIL Research, supplemented Dr. Robert Parker’s discussion of juvenile toxicity studies with special considerations in bio-pharmaceutical drug development. Biopharmaceuticals have unique characteristics that can change traditional study designs and have specific challenges such as structural and biologic diversity, species specificity, and immunogenic potential (Morford et al. 2011). Pharmacological relevance must be established in the test species and species specificity often limits applicability of on- and off-target effects to the nonhuman primate (NHP). Immunogenicity can significantly impact the design and interpretation of animal studies but does not reliably predict immunogenicity in humans (Morford et al. 2011). Dr. Morford discussed the advantages and disadvantages of using surrogate molecules, genetically engineered mouse (GEM) models, and various species including NHPs, rodents, dogs, and minipigs in juvenile toxicity studies for biopharmaceuticals. While the rat remains the preferred species in nonclinical evaluation for pediatric safety assessment of small molecules, NHPs are often the only applicable species for biopharmaceuticals. However, variation in biopharmaceutical properties drives the need to find the most relevant animal model on a case-by-case basis, if a relevant model is available.

Pathology Perspectives

In the afternoon session, the meeting’s focus shifted to the pathology perspectives of juvenile toxicity testing. George A. Parker, DVM, PhD, DACVP, DABT, vice president of Global Pathology at WIL Research, reviewed the major challenges faced by pathologists in the assessment and interpretation of histopathological changes in tissues from juvenile animals. Dr. George Parker discussed the dynamics of postnatal development, using the immune system as the primary example (G. A. Parker et al. in press). Significant tissue changes occur during the course of a juvenile toxicity study. The biggest challenge faced by pathologists in assessing juvenile toxicity studies is the absence of concurrent controls when animals die or are euthanized prior to the scheduled study endpoint. This issue highlights the importance of the pathologist’s need to understand normal tissue development at different ages in light of the typical relative lack of experience with juvenile tissues and the lack of concurrent controls for comparison. To address this point, Dr. George Parker proposed adding more non-dosed control animals to a given study to serve as age and sex-matched controls for unscheduled deaths. This would also enable pathologists to accumulate a database representing the different time points that could be used as a reference for future studies.

Catherine A. Picut, VMD, JD, DACVP, DABT, senior pathologist with WIL Research, presented the beginnings of a project to publish a detailed atlas of juvenile histology. Dr. Picut’s presentation of this project served as an appropriate and relevant illustration of the database of information that could be accumulated on juvenile tissues and used as a reference for pathologists. Dr. Picut reminded the audience that while we generally refer to “juvenile” toxicology, this subject truly encompasses multiple early life stages from neonatal, to early and late infantile, juvenile, and peripubertal. In the evaluation of juvenile toxicity endpoints, it is important for pathologists to recognize normal development from abnormal development through all these life stages. The testis, ovary, thyroid, brain, and lung were used as example tissues to demonstrate the dynamic fluctuations and salient histological developmental landmarks that occur throughout postnatal development (Bandeira, Lent, and Herculano-Houzel 2009; Bayer 1982; Bayer et al. 1993; Downes and Mullins 2014; O’Reilly and Thebaud 2014; Picut et al. 2014; Picut et al. 2015a; Picut et al. 2015b; Schittny and Burri 2008).

Focusing on the kidney, John C. Seely, DVM, DACVP, senior pathologist at Experimental Pathology Laboratories, Inc., described the toxicologic and pathologic issues surrounding juvenile renal studies. The importance of not only morphologic development but also functional development of the kidney was highlighted. Completion of morphologic nephrogenesis occurs around PND 8 to 11 while full functional renal maturation occurs by 6 to 7 weeks of age in the rat (Zoetis and Hurtt 2003a). The current stage of nephrogenesis and functional maturation of the kidney with variations in parameters such as renal blood flow, drug transport systems, and clearance mechanisms will result in striking differences in drug sensitivity (Schreuder et al. 2011). In most cases, the developing kidney appears to be more resistant than the adult to toxic agents; thus, juvenile nephrotoxicity cannot be extrapolated from adult data (Cappon and Hurtt 2010). The pathologist must be acutely aware of these potential differences for accurate interpretation of study data.

In addition to understanding differences in organ development within a species, the pathologist must be aware of differences in development between species. Using the NHP as an animal model in juvenile studies can add some significant challenges. However, as Dr. Morford indicated in her earlier discussion, in certain situations the NHP is the only relevant animal model. J. Mark Cline, DVM, PhD, DACVP, professor of pathology/comparative medicine and head of the section on comparative medicine at Wake Forest School of Medicine, delved into the pros and cons of using NHPs in toxicity studies and how one can manage some of the challenges. While NHPs have genetic and physiological similarities to humans, the ethical concerns, expense, biohazard potential, and high individual variability make sound scientific justification and appropriate handling of this species a must. With careful planning and design of studies using NHPs, and objective documentation of age and sexual maturation status especially when using juvenile animals, the interindividual animal variability can be partially controlled. Controlling this variability will aid in more accurate interpretation of subtle test article–related effects and yield more reliable results needed for extrapolation of NHP data to humans (Chellman et al. 2009; Cline and Wood 2008; Mansfield and Kemnitz 2008; Mattison et al. 2011; Morton et al. 2008; Rodriguez et al. 2010; Sasseville and Diters 2008; Schoeb et al. 2008; Stute et al. 2012; Tharp et al. 2012; Uckun et al. 1997; Yasuda et al. 2005).

The meeting was concluded with a summary of key points from each presenter given by Dr. Remick. Final closing remarks by Dr. Dixon thanked the participating speakers and audience members for a successful and highly informative regional meeting on the relevance and challenges of juvenile toxicology for toxicologists and pathologists.

Abbreviations

BCPA

Best Pharmaceuticals for Children Act

CDER

Center for Drug Evaluation and Research

DABT

Diplomate of the American Board of Toxicology

DACVP

Diplomate of the American College of Veterinary Pathologists

DVM

Doctor of Veterinary Medicine

FDA

Food and Drug Administration

FDAAA

FDA Amendments Act

FDASIA

FDA Safety and Innovation Act

GEM

genetically engineered mouse

JD

Juris Doctor

MOG

modified one-generation

NHP

nonhuman primate

NIEHS

National Institute of Environmental Health Sciences

NTP

National Toxicology Program

NTPL

National Toxicology Program Laboratory

PND

postnatal day

PREA

Pediatric Research Equity Act

RACB

Reproductive Assessment by Continuous Breeding

STP

Society of Toxicologic Pathology

VMD

Veterinariae Medicinae Doctoris (equivalent to DVM)