Evaluation of Juvenile Animal Studies for Pediatric CNS-Targeted Compounds: A Regulatory Perspective

Abstract

Central nervous system (CNS)-targeted products are an important category of pediatric pharmaceuticals. In view of the significant postnatal maturation of the CNS, juvenile animal studies (JAS) are performed to support pediatric development of these new medicines. In this project, the design and results of juvenile toxicity studies from 15 drug compounds for the treatment of neurologic or psychiatric conditions were analyzed. Studies were conducted mostly in rats; sometimes in addition in dogs and monkeys. The study design of the pivotal JAS was variable, even for compounds with a similar therapeutic indication. Age of the juvenile animals was not consistently related to the starting age of the intended patient population. Of 15 compounds analyzed, 6 JAS detected more severe toxicities and 6 JAS evidenced novel CNS effects compared to their adult counterparts. The effects of CNS on acoustic startle and learning and memory were observed at high dosages. Reversibility was tested in most cases and revealed some small effects that were retained or only uncovered after termination of treatment. The interpretation of the relevance of these findings was often hampered by the lack of matching end points in the adult studies or inappropriate study designs. Detailed clinical observation and motor activity measures were the most powerful end points to detect juvenile CNS effects. The need for more detailed behavioral examinations in JAS, for example, on learning and memory, should, therefore, be decided upon on a case-by-case basis, based on specific concerns in order to avoid overloading the studies.

Introduction

With an increasing requirement for specific safety assessment of medicines for the pediatric population, regulatory authorities have published guidelines to be consulted in case of development of pharmaceuticals for use in the pediatric age range. ^1,2 In various regions of the world, this has led to a new regulatory branch of safety testing, for example, nonclinical safety testing in juvenile animals. ^3,4 Initially, a specific regulatory framework regarding the design of toxicology studies to support pediatric development was lacking. It was not clear what type of outcomes could be expected from the studies in juveniles, that is, a difference in targets of toxicity and/or a difference in sensitivity in juveniles compared to adults. Such differences might be due to organ/system immaturity or effects on organ/system development. ^3,4

One area of concern is the central nervous system (CNS), as children might benefit from treatment through improvement of their neurological condition, but at the same time could suffer from adverse effects on the CNS, such as sedation or effects on cognition.

A high number of pharmaceuticals targeting the CNS are on the market. Because of signals that psychotropic drugs can have negative effects on cognition, it was discussed that for these CNS-targeted drugs relevant information could be gained from safety studies in young animals. ⁴ These studies may contribute to predicting whether the exposure to a drug in pediatric patients can lead to developmental and/or functional abnormalities and to identifying toxicological signals and/or possible biomarkers that are not seen in adults. ^2,5,6

It is worthwhile to systematically evaluate the outcome of these studies. The aim of this European Union (EU) analysis is to evaluate juvenile animal safety data with respect to study design, end points (particularly those expected to detect neurological side effects), and results and to learn from juvenile toxicity approaches for CNS compounds. The outcome of this analysis may also be important for the discussion of the International Council for Harmonisation (ICH) regarding guidance on nonclinical safety testing in support of the development of pediatric medicines. ⁷

Methods

Product Selection

The virtual archives of the Pediatric Committee (PDCO) at the European Medicines Agency (EMA) were searched for data regarding compounds with a Pediatric Investigation Plan (PIP) in therapeutic areas of neurology, psychiatry, anesthesiology, and pain. Only those directly targeting the CNS (ie, CNS-targeted compounds) for which juvenile animal studies (JAS) were performed and/or requested for were considered for inclusion in the analysis. Data were available for PIPs which were agreed between 2008 and 2015, that is, between the coming into force of the EU Paediatric Regulation and the date of the analysis. ⁸ Complete data packages consisted of PIP summary reports for initial and modification procedures, the investigator brochure, and toxicology study reports both in juvenile and adult animals. The regulatory impact was determined by checking the PDCO assessment reports, and the decisions of the Committee for Medicinal Products for Human Use (CHMP) reflected in the Summary of Product Characteristics (SmPC).

A total of 83 compounds were retrieved from the EMA database in therapeutic areas of neurology, psychiatry, anesthesiology, and pain. Of these, 58 compounds were CNS-targeted and only 25 of these compounds JAS reports were available. Only 15 compounds corresponding adult nonclinical safety data were available and only those compounds were selected for further analysis. Indications targeted by compounds with available complete data packages were De la Tourette and restless legs syndrome (Tou), narcolepsy (Nar), schizophrenia (Schi), attention-deficit hyperactivity disorder (Adhd), depressive and anxiety disorders (Dep), and epilepsy and bipolar I disorders (Epi; Table 1). These compounds are anonymized to protect commercial interests using an alphanumerical code related to the target indication with the abbreviation as indicated.

Table 1.

Characterization of the Compounds Assessed (Pharmacology, Therapeutic Condition, and Age Range).

Compounda	Pharmacology	Condition	Pediatric age range (years) in scope of pediatric investigation plan
Adhd1	Nonstimulant α2A adrenergic receptor agonist	Attention-deficit hyperactivity disorder (ADHD)	6 < 18
Adhd2	Increasing catecholamine release at synaptic region	ADHD	6 < 18
Dep1	5HT transporter inhibitor, 5HT7, 5HT3, and 5HT1D antagonist, 5HT1B partial agonist, and 5HT1A agonist.	Major depressive disorder	7 < 18
Dep2	Melatonin agonist with high affinity for MT1 and MT2 receptors. Also 5HT2C antagonist. Metabolites 3HD and 7DP have moderate activity.	Major depressive disorder	7 < 18
Epi1	Voltage-dependent sodium channel inhibitor	Lennox-Gastaut syndrome	1 < 18
Epi2	Confidential information	Epilepsy syndromes	Birth (preterm) < 18
Epi3	Voltage-dependent sodium channel inhibitor	Epilepsy with partial-onset seizures	2 < 18
Epi4	High affinity for synaptic vesicle protein 2A also checked for Na+ voltage-gated channels, but not positive	Epilepsy with partial-onset seizures Neonatal seizures	Birth (preterm) < 18
Epi5	Noncompetitive antagonist at AMPA glutamate receptor	Treatment-resistant epilepsies	Birth (preterm) < 18
Epi6	Facilitates opening of K+ channels	Epilepsy with partial-onset seizures Lennox-Gastaut syndrome	Birth (preterm) < 18
Nar1	Wake promoting compound	Narcolepsy	6 < 18
Schi1	Antagonist of D2 and 5HT2a receptors. Also affinity for α1, H1 and α2 receptors. Not bound to cholinergic receptors.	Schizophrenia	12 <18
Schi2	Combination of D2 and 5HT2 antagonist also affinity for 5HT1A, 5HT1b, 5HT2c, 5-HT6, 5HT7, D3, and α2 receptors	Bipolar I disorder	10 < 18
Schi3	Partial agonist at D2 and 5HT1a receptors, antagonist at 5HT2a receptors	Schizophrenia Bipolar I disorder	10 < 18
Tou1	Full agonist on D2 with subtypes D2, D3, and D4	Combined motor and multiple motor tic disorder (De la Tourette) Restless leg syndrome	6 < 18

^aCompounds are anonymized in association with the intended indication: Adhd, attention-deficit hyperactivity disorder; Dep, depressive and anxiety disorders; Epi, epilepsy and bipolar I disorders; Nar, narcolepsy; Schi, schizophrenia; Tou, de la Tourette and restless legs syndrome.

Data Evaluation

Once all available data were retrieved, an evaluation form was developed and used to make a full assessment of the juvenile animal data for the selected compounds. The following parameters were summarized and assessed for every compound and each juvenile toxicity study performed: clinical indication, youngest age of the patient population, intended human dose and exposure at this dose, species selection, age of animals at study start, study duration, dosing regimen in animal study, timing of CNS end points in relation to time of dosing as well as day of the study, animal exposure end points (area under the curve for 0-24 hours [AUC_0-24]; C_max, at different time points, if available), and main treatment-related effects. For each compound, an adult repeated-dose study in the same species was also assessed to directly compare the exposures and effects with the respective JAS, at similar doses and treatment duration, where available. In addition, the overall pharmacotoxicological data package was used to interpret the findings in relation to each compound’s mechanism of action (pharmacology) and to the effects in adult animals. Finally, regulatory evaluation and resulting inclusion of the information into the regulatory documentation by the EMA’s CHMP were assessed.

Results

Species Selection and Number of Animals

Of the 15 compounds rats were used in JAS with 14 out of these. A second species was used in 7 cases (dogs in 6 cases [Adhd2, Epi1, Epi2, Epi4, Epi5, Schi3], Rhesus monkeys in one case [Tou1]). For the remaining compound (Epi3), the dog was the single species used. For one case (Epi6) in which initially mice had been used, the mouse data were discarded in view of excipient toxicity (see later), and rat data were generated using another excipient.

The number of animals in the pivotal studies in rats and nonrodents is given in Table 2. The number of rats used per study varied between 250 and 760, and in one case up to 1,120 pups. Rats used for toxicokinetic (TK) assessment are included in these numbers. For nonrodents, the number of animals used per study is also variable and varied between 47 and 140.

Table 2.

Study Design Elements of the Juvenile Animal Studies Assessed.

Compounda	Pediatric age range (years) in scope of pediatric investigation plan	Starting age	Route of administration	Treatment/recovery (weeks or month)	Species	Number of juvenile animals in pivotal JAS
Adhd1	6 < 18	PND 7	PO	13 weeks/4 weeks	Rat	252
	6 < 18	PND 7	PO	7.5 weeks/no rec.	Rat	256
Adhd2	6 < 18	PND 7	PO	8 weeks/4 weeks	Rat	552
		PNW 10	PO	26 weeks/4 weeks	Dog	64
Dep1	7 < 18	PND 21	PO	10 weeks/4 weeks	Rat	352
Dep2	7 < 18	PND 7	PO	10 weeks/4 weeks	Rat	528
Epi1	1 < 18	PND 7	PO	10 weeks/4 weeks	Rat	580
		PND 42	PO	14 weeks/4 weeks	Dog	47
Epi2	Birth (preterm) < 18	PND 12	PO	5 weeks/2 weeks	Rat	336
		PND 70	PO	26 weeks/4 weeks	Dog	48
Epi3	2 < 18	PND 21	PO	10 months/2 months	Dog	97
Epi4	Birth (preterm) < 18	PND 4	PO	9 weeks/4 weeks	Rat	1,120
		PND 4	PO	9 months/2 months	Dog	140
Epi5	Birth (preterm) < 18	PND 7	PO	12 weeks/4 weeks	Rat	684
		PND 42	PO	33 weeks/4 weeks	Dog	64
Epi6	Birth (preterm) < 18	PND 28	PO	12 weeks/4 weeks	Rat	446
Nar1	6 < 18	PND 7	IP	9 weeks/3 weeks	Rat	760
Schi1	12 < 18	PND 24	PO	7 weeks/4 weeks	Rat	256
Schi2	10 < 18	PND 14	SC	8 weeks/months: 6-f: 8 weeks	Rat	320
Schi3	10 < 18	PND 21	PO	2 months/0 weeks	Rat	544
		PNM 2	PO	6 months/2 months	Dog	48
Tou1	6 < 18	PND 21	PO	7 weeks/4 weeks	Rat	464
		PNM 20-24	PO	30 weeks/12 weeks	Monkey	56

Abbreviations: IP, intraperitoneally; JAS, juvenile animal studies; PND, postnatal day; PNM, postnatal month; PO, orally; SC, subcutaneously.

^aCompounds are anonymized in association with the intended indication: Adhd, attention-deficit hyperactivity disorder; Dep, depressive- and anxiety disorders; Epi, epilepsy and bipolar I disorders; Nar, narcolepsy; Schi, schizophrenia; Tou, De la Tourette and restless legs syndrome.

Starting Age

When comparing the age of the animals at study start (Table 2) with the youngest age of the intended patient population, a major heterogeneity was observed. The 6 antiepileptic compounds, most being developed to treat neonates including preterm newborns, showed significant differences in starting age. In rats, it ranged from postnatal day (PND) 4 to PND 28, in dogs from PND 4 to PND 70.

For a product for the treatment of Tourette syndrome, which can be diagnosed in patients from 6 years of age, rats PND 21 were studied. For the 2 antidepressants, with a similar patient age range, the rat JAS started at PND 7 and PND 21. Although the youngest target age for Adhd and Nar was also 6 years, the JAS for these products all started at PND 7 in rats. The 3 compounds to treat schizoaffective disorders and developed for children aged from 10 to 12 years had ages at study start in rats varying from PND 14 to PND 24.

Duration of Treatment and Recovery

The duration of treatment varied from 5 to 13 weeks in rats. For Epi2, the treatment lasted only up to PND 46, whereas for Epi6 it lasted until PND 112. Most studies lasted until PND 70 with a variance up to PND 91. This is in-line with guideline recommendations to treat animals up to reaching adulthood when the target organs have a long development period ^2,3 and also takes into account that most of the compounds are intended to be used chronically.

Route and Frequency of Administration

In nearly all studies, daily administration by the oral route was applied in-line with the intended clinical route. For Nar1, the oral route was tried initially but did not result in a clinically relevant level of exposure. Therefore, the intraperitoneal (IP) route was applied in the juvenile rat study to enhance exposure. For Schi2, the subcutaneous route was used in toxicity studies as relevant to the clinical route (sublingual). For Nar1, Epi4, and Dep1, compounds were administered twice a day (BID administration). Of note, when oral BID administration started in very young rats (PND 4, Epi4), higher than usual mortality was noted mainly before weaning in all groups, including controls (19, 9, 20, or 69 unscheduled deaths at 0, 150, 300, or 600 mg/kg, respectively).

Use of Excipients

In the early studies with Epi6, propylene glycol was used as excipient (5 mL/kg of body weight), which led to mortality at all doses including controls in the juvenile animals of choice (in this case, mice). These mouse studies were discarded in the evaluation because of this confounding mortality. Later on, rats were tested with hydroxymethylcellulose to aid a stable suspension and these data were used for the current evaluation.

Exposure: Comparison Between Juvenile and Adult Animals

All cases were selected to have adequate determination of systemic exposure in the pivotal JAS. Maximum concentration (C_max) was given and the AUC during 24 hours (AUC_0-24) had been calculated by the sponsor. In a majority of the JAS, blood sampling to determine plasma levels of the test article was performed on 2 occasions: close after study start and at the end of the study. Sometimes, an additional sampling was included around the time of weaning. We have chosen to focus on the AUC_0-24 as a measure to compare exposure between juvenile and adult animals, although it is acknowledged that in some cases, the C_max would represent a more relevant measure of exposure in relation to specific CNS effects. For some compounds, exposure in adult animals had not been determined, and in those cases the “adult exposure data” were taken from the JAS latest time point, usually from young adult animals. Brain concentrations were not determined in any of the reviewed studies.

In Table 3 (third column), the detailed outcome of the comparison between juvenile and adult animals is presented for all individual compounds. Generally, in rats, when treatment was started preweaning (PNDs 7-14), exposure was initially high, followed by a decrease around weaning time or even up to PND 35, while increasing again at later ages, although not as high as in the preweaning period (eg, Epi1 and Epi5). Differences in exposure were usually only 1.5- to 3-fold. When administration started around or just after the time of weaning, the exposure was usually found to increase toward the end of the treatment period (PNDs 70-98).

Table 3.

Comparison of Exposure and Toxicity in Juvenile and Adult Animals in the CNS Compounds That Were Assessed.

Compounda intended age	Starting age	Comparison of toxicity	Conclusion
Adhd16-18 years	Rats PND 7	Juvenile rats: NOAEL: 0.3 mg/kg due to the effects on body weight gain and abnormal behavioral observations observed at higher doses. AUC0-tlast: PND 13: 11 ng h/mL, PND 96: 5 ng h/mL 3 mg/kg: Behavioral effects (increased agitation, tenseness, vocalization, and aggressiveness, followed by decreased activity/no movement/fixed gaze) observed at the end of the dosing period (≥PND 80). Not observed during the reversibility period. AUC0-tlast: PND 13: 310 ng h/mL, PND 96: 131 ng h/mL Adult rats: Dose-related incidence of corneal opacities (considered to be related to the α-adrenergic activity of Adhd1), not observed in juvenile rats. NOAEL: 1 mg/kg Higher doses: Effects on body weight and food consumption and wall thickening of the small and large intestines of rats. No TK data available.	At the same dose levels, exposure at PND 13 was ∼2-fold higher compared to PND 96 animals (JAS). Novel toxicities: Behavioral effects starting on PND 80 in juvenile animals. Not observed during the reversibility period or in the repeated-dose toxicity study. No apparent increased sensitivity in juvenile animals. No comprehensive neuropathology evaluation reported.
Adhd26-18 years	Rats PND 7Dogs week 10	Adhd2 is the inactive prodrug of its major active metabolite. Juvenile rats: >NOAEL: 4 mg/kg: Increased activity AUC0-24 of metabolite A: PND 7: 603 ng h/mL, m/f PND 63: 281 ng h/mL, m/f >10 mg/kg body weight, food consumption, and growth measurements changes ≥40 mg/kg: Reproductive effects attributed to effects on body weight Adult rats: NOAEL (lowest dose) 20 mg/kg: Similar findings as in juvenile animals AUC0-24 of Adhd2: Study day 1 (∼PND 49): 907 ng h/mL Study day 28: 883/2,818 ng h/mL, m/f Juvenile dogs ≥2 mg/kg: Postdosing increased activity, stereotypic behavior, nonresponsiveness, and/or vocalization AUC0-24 of Adhd2: Study day 1: 212 ng h/mL 6-Month: 445 ng h/mL ≥5 mg/kg: Body weight, food consumption, and urine volume effects increased incidence of reduced activity and muscle tremors. AUC0-24 of Adhd2: Study day 1: 628 ng h/mL 6-Month: 1,151 ng h/mL Adult dogs: >NOAEL: 2 mg/kg (no exposure data available) Pharmacology-driven dose-related effects on body weight and behavior	Exposure decreased about 2-fold from PND 7 to PND 63 in juvenile rats and increased about 1.5-fold from study day 1 to study days 182 to 183 in juvenile dogs Effects which were identified in adults and juvenile animals were mostly related to the pharmacology of the major metabolite. No new toxicities or exacerbate effects were identified in juvenile animals compared to adult animals in the rats. In juvenile rats, neuropathology evaluations of peripheral nervous system and CNS (H&E staining) did not raise signs of neurotoxicity.
Dep17-18 years	Rat PND 21	Both juvenile and adults: BID administration Juvenile rats: Nondose-related increased motor activity scores observed at all doses >10 mg/kg: Salivation and paddling of the forelimbs during treatment and decreased body weight gain during treatment-free period. NOAEL: 20 mg/kg In addition: increased prothrombin time, liver changes (weight and hepatocellular hypertrophy), and decreased salivary gland weight AUC0-10 h nmol h/mL, m/f: PND 21: 1,017/935, PND 91: 4,188/3,593 40 mg/kg in addition: Corticomedullary mineralization in the kidney and adverse effect on the survival and clinical condition of the offspring of those parents previously given 40 mg/kg BID which cannot be ruled out as treatment related. Adult rats: ≥20 mg/kg (NOAEL in males): Occasional salivation and increased body weight gain (attributed to increase in food conversion efficiency) during treatment, followed by lower body weight gain during treatment-free period in females only AUC0-10 h nmol h/mL, m/f: Study day 1: 1,235/1,672, study week 13: 4,778/5,029 40 mg/kg (NOAEL for females): in addition: Kidney and liver changes, hematology, and clinical chemistry effects AUC0-10 h nmol h/mL, m/f: Study day 1: 3,508/4,551, study week 13: 17,860/14,165	Age and/or plasma accumulation-related increases in exposure were noted from PND 21 up to PND 91 (∼4-fold) during the JAS and from study day 1 up to week 13 during the adult study (∼3.5-fold). Exposure on study day 1 of the adult study is about 1.5 higher than exposure on PND 21 (JAS). All treatment-related findings observed in juvenile rats were consistent with those noted in adult animals, with the exception of an apparent adverse effect on the survival and clinical condition of the offspring of those parents previously given the highest dose (40 mg/kg BID) which cannot be ruled out as treatment related (increased incidence of empty stomachs without effect on body weight). No comprehensive neuropathology evaluation reported.
Dep27-18 years	Rat PND 7	NOAEL in juveniles 5-fold lower than in adults. Not fully clear what constitutes the NOAEL. Juvenile rats: 5 mg/kg (female NOAEL) Females: Decreased body weight gain limited to the preweaning period. However at weaning, there were no significant differences in body weight compared to controls. Males: Decreased body weight gain was also noted postweaning. Effects on reproductive organs and decrease in absolute and/or relative (to body and/or brain) weight of the pituitary (not reversible during the recovery period) Based on these effects observed at the lower dose, a NOEL was not determined for males. AUC at 5 mg/kg: PND 7: 4,887(m), 5,107(f) ng h/mL, PND 76: 115(m), 157(f) ng h/mL >30 mg/kg: Effects on body weight, growth, and reproductive organs. Decreased pituitary, prostate, testes, and uterus with histological changes. During the preweaning treatment phase (up to PNDs 14-15), decreased activity, decreased respiratory rate, and also uncoordination and subsequently lying on sides were noted. AUC at 30 mg/kg: PND 7: 48,794(m), 45,367(f) ng h/mL, PND 76: 1,683(m), 2,281(f) ng h/mL Behavioral tests and reproductive performance were unaffected up to 150 mg/kg. Adult rats: 25 mg/kg: NOAEL both genders Slight effect on body weight in males AUC (ng h/mL): Study week 14: 2,394, study week 26: 2,407 ≥125 mg/kg: Effect on heme synthesis and associated urine changes 625 mg/kg: Decreased spontaneous activity, effects on body weight and water consumption, and moderate prolongation of clotting time Liver and thyroid changes were attributed to enzyme induction.	In juvenile rats exposure at PND 7 much higher than at PND 76 (37-fold). Female exposure at NOAEL was 2.2-fold higher in juvenile rats on PND 7 than in adults on week 14 but 16-fold lower on PND 76 compared to week 26 in adults. Novel target toxicity mainly related to growth end points (long bone measurements) and organ weights particularly pituitary weight (absolute and relative to body weight) decreased during the treatment and off-treatment phases. In juvenile rats, effects on body weight and clinical observations were noted primarily during the preweaning period consistent with higher exposures during this phase of the study. In adult rats, similar effects on body weight were seen from the lower dose. As in juveniles, males seem more sensitive than females. Comparison of toxicity profiles between juvenile and adult rats is hampered by the very early start of the juvenile study (with higher exposure levels) of the juvenile study (human use starts in 7-year-old children) and the much higher exposure levels achieved in adult rats at the high doses. No comprehensive neuropathology evaluation reported.
Epi11-18 years	PND 7	Juvenile rats: NOEL: 15 mg/kg (239, 119, 147 AUC µg h/mL on PNDs 7, 21, and 147, respectively) LOAEL: 50 mg/kg (588, 331, 447 µg h/mL on PNDs 7, 21, and 147, respectively) lower absolute brain weight (relative brain weight did not change, but following fixation, the brain and region of cerebrum were significantly reduced). Lower thymus weight and higher testis weights were noted. Cytoplasmic vacuolation was observed in pituitary in male animals. Adult rat study (started at PND 42) was conducted with much higher dosages (200 up to 1,000 mg/kg). NOAEL could not be established. AUC measured in juvenile rats given 150 mg/kg (highest dose) was of the same order of magnitude as in adult rats given 200 mg/kg (lowest dose) Juvenile dogs: 20 mg/kg biliary pigments were observed in centrilobular bile canaliculi. Doses and exposure in adult dogs treated for 26 of 52 weeks were the same as in juvenile animals.	In juvenile rats, higher exposure was recorded on PND 7, which decreased ∼2-fold up to PND 21 and increased again up to adulthood. In dogs, no differences have been found between exposure of juvenile and adult dogs. Existing juvenile rat and dog data did not indicate that juvenile animals are more sensitive than the adult animals. With dosages >50 mg/kg, lower brain weight was found in rats. Further histopathological evaluations (specific staining aimed at detecting signs of neurotoxicity) did not raise any concern.
Epi20-18 years	Rat PND 12Dog PND 70	Juvenile rats: NOEL: 20 mg/kg (AUC at PND 18 of (m/f) 49.4/51.9 µg h/mL) LOAEL: 80 mg/kg (AUC 217/224 µg h/mL) hypotonia, coldness, tremors, piloerection, hypokinesia, and staggering gait. Also decreased body weight, increased bilirubin, and cholesterol levels. Lethal dose: 240 mg/kg (dose range finding study) Adult rats: NOAEL: 30 mg/kg, AUC (m/f) 108/130 µg h/mL LOAEL: 100 mg/kg, AUC m/f 347/361 µg h/mL, small effects on diuresis, increased liver weight increased cholesterol and bilirubin, and lower AST levels. No CNS symptoms mentioned. Juvenile dogs: NOAEL: 30 mg/kg PO, AUC on PND 70 of 41.7 µg h/mL increasing up to 87.4 µg h/mL on PND 252, observed effects: underactivity, unsteady/abnormal gait, and body tremors, also at lower doses. LOAEL: 60 mg/kg, AUC of 108 µg h/mL increasing up to 168 µg h/mL on PND 252, similar effects and decrease in body weight (17%) with reversal in 4-week recovery period. Adults dogs: NOAEL: 20 mg/kg, AUC 129 µg h/mL on study day 24, abnormal stool, decreased activity, and abnormal gait or stance.	Lower exposure (∼2- to 3-fold) in juvenile rats and dogs versus adults at similar doses. No new toxicity. In rats, juveniles were more sensitive to CNS clinical signs compared to adult rats. This is based on the observation of acute lethality, likely linked to exaggerated pharmacodynamic effects, observed at the high dose (240 mg/kg, study days 1-2), showing no similar (lethal) effect in adult rat studies (up to 300 mg/kg, administered for 3 months). The higher sensitivity cannot be explained by increased systemic exposure or accumulation. Brain concentrations were not determined. In dogs, LOAEL AUC in juveniles is similar to NOAEL in adult animals. Juveniles thus appeared to be more sensitive. Effects on body weight are more pronounced.
Epi32-18 years	Dogs PND 21	Administration of Epi3 to dogs and humans give rise to 2 active metabolites (A and B). These are enantiomers. The major one (A) is reported in juvenile animals (exposure to metabolite B has been limited to a few time points in dogs from the high-dose group), the racemic mixture (A+B) in adult dogs: Juvenile dogs: NOAEL: 40 mg/kg LOAEL: 80 mg/kg ≥40 mg/kg: Higher serum cholesterol, triglycerides, alkaline phosphatase, and γ-glutamyltransferase levels—no organ weight or microscopic correlates AUC0.5-24 h: 4,045 ng h/mL on PND 21 and 1,167 ng h/mL on PND 328 >80 mg/kg: Mortality, linked to dose-related decreased activity, hypoactivity, impaired muscle coordination, rigid muscle tone, impaired equilibrium, tremors, convulsions, vocalization, labored respiration, pale body cool to touch, dermal atonia, injected sclera of the eye(s), nystagmus, excess salivation, clear and/or white frothy material around mouth, emesis, and/or pale gums. Reversible lower bone mineral content, area, and density in the femurs and lumbar vertebrae (L3-L5)—no serum chemistry or microscopic correlates. AUC0.5-24 h: 7,795 ng h/mL on PND 21, 1,880 ng h/mL on PND 328 Adults dogs: NOAEL: 40 mg/kg: Sporadic pre- and postdose salivation. Reduced body weight gain and increased cholesterol levels AUC0-24: 2,057 ng h/mL on study day 1, 1,530 ng h/mL on study week 52 LOAEL: 80 mg/kg: Pre-and postdose salivation almost daily, subdued behavior or abnormal gait, loose feces, vomiting, and tremors. Reduced body weight gain, reduced food consumption in males, increase in cholesterol levels, and increase in mean relative liver weight AUC0-24: 4,467 ng h/mL on study day 1, 3,707 ng h/mL on study week 52,210 mg/kg: Dose down to 160 mg/kg due to severity of the effects (2 dogs sacrificed due to marked clinical signs): subdued behavior, head shaking, vomiting, abnormal and/or unsteady gait, muscular rigidity, lip licking, and lateral recumbency and unresponsive behavior (1 male).	No marked difference in exposure to the active metabolites between juvenile and adult dogs. Exposure tends to decrease with duration of the study at both ages (3.5-to 4-fold in juvenile dogs). New toxicity: bone effects (no mineral density assessed in adult dogs). The toxicity profile in juvenile dogs was comparable to that observed in adult animals. Nevertheless, juvenile dogs seem more responsive to CNS side effects as mortality associated with severe effects on behavior were observed at LOAEL (80 mg/kg) in JAS, while in adult dogs at the same dose, CNS effects were less pronounced and no death was observed. CNS and peripheral nervous system tissues were submitted to microscopic neuropathological examination from an extensive set of tissues using specific staining aimed at detecting signs of neurotoxicity. None were detected.
Epi40-18 years	Rat PND 4Dog PND 4	BID drug administration in both rats and dogs Juvenile rats: NOAEL: 300 mg/kg: Reversible hepatocellular hypertrophy consistent with higher liver weights and increased hyaline droplets in the proximal convoluted tubules of the kidneys AUC0-24 h: 168 µg h/mL on PND 21, 373 µg h/mL on PND 70 LOAEL: 600 mg/kg: Increased mortality (occurring mainly between PND 11 and PND 21) compared to control, low- and mid-dose groups (19, 9, 20, or 69 unscheduled deaths on 0, 150, 300, or 600 mg/kg, respectively). These excess deaths were considered related to treatment with the test item. Clinical signs for these animals consisted of pale and/or cool bodies, gasping, labored respiration, rales, hypoactivity, slightly drooping or completely shut eyelids, and/or rocking, lurching, or swaying while ambulating. Lowered body weight partially recovered by weaning Isolated effects on motor activity, startle response Statistically lower absolute and relative brain weights. Effects on the liver and lungs as with lower dose. AUC0-24 h: 226 µg h/mL on PND 21, 583 µg h/mL on PND 70 Adult rats: Neither CNS effects nor test article-related deaths were recorded up to 400 mg/kg dose in adults. Increased liver weight and hypertrophy of centrilobular hepatocytes observed at all doses-considered adaptive response. Lipofuscine (brown pigments) and multilamellar structures in centrilobular hepatocytes at 200 and 400 mg/kg. NOAELs: 50 mg/kg in females (AUC 133 µg h/mL, day 1) and 100 mg/kg in males (AUC 175 µg h/mL, day 1) 400 mg/kg: Changes of small amplitude in blood chemistry (eg, alanine aminotransferase, not totally reversible in high-dose females) and hematology parameters Juvenile dogs: NOEL: 15 mg/kg AUC0-24: 85.3 µg h/mL at PND 4, decreasing to 28.4 at PND 31 and 37.1 at PND 276 100 mg/kg: More outspoken liver toxicity, decreased T4 level, and thymus weight Adults dogs: NOAEL: 37.5 mg/kg with increased liver weight and decreased thyroid weights AUC: 104 µg h/mL	In juvenile rats, exposure increased from PND 21 to PND 70 (∼2.5-fold) to reach the same order of magnitude as in adults at similar dose levels. In juvenile dogs, exposure levels (AUC) are higher on PND 4 than PND 31 and 276 (∼2- to 3-fold). Novel toxicities: In juvenile rats, at the highest dose level, mortality and effects on startle response, motor activity, and decreased absolute brain weight were observed (not observed in adults). Brain weight effects were likely related to changes in general body weight. Seemingly increased sensitivity of juvenile rats since test article-related death was recorded at 600 mg/kg in juveniles (AUC0-24h: 226 µg h/mL, PND 21, 583 µg h/mL, PND 70), but not in adults given 400 mg/kg (AUC0-12 h 778 µg h/mL, study day 1)—this may be due to higher exposure levels observed in rats at PND 4 (AUC0-24 h: 2,349 µg h/mL). In juvenile rats, CNS and peripheral nervous system tissues were submitted to microscopic neuropathological examination from an extensive set of tissues using H&E staining. No treatment effects were detected.
Epi50-18 years	Rat PND 7Dog PND 42	Juvenile rats: ≥1 mg/kg (NOAEL): Pharmacologically mediated clinical signs (decreased activity, incoordination and lying on side during lactation period, and decreased activity, uncoordination, excessive grooming/licking/scratching, and ptosis after weaning were observed at all doses) AUC0-24: PND 7: 2,180/2,009, PND 35: 564/1,127, PND 63: 739/1,584, PND 90: 869/1,620 ng h/mL (m/f) >3 mg/kg: Secondary to these signs, reduction in growth progression was observed (body weights, crown-to-rump lengths, femur and tibia lengths, delayed preputial separation, and vaginal opening) AUC0-24: PND 7: 6,062/6,409, PND 35: 2,105/1,946 PND 63: 1,451/2,678, PND 90: 1,676/3,018 ng h/mL (m/f) • Adult rats: Treatment effects limited to pharmacologically based clinical signs consisting of abnormal gait, decreased activity, sedation, and prostration. 1 mg/kg: NOAEL in female AUC 852 ng h/mL, PND 56 10 mg/kg: NOAEL in male In females: Decreased food consumption, abnormal gait, decreased activity, prostration, and mydriasis. AUC: 3,125 ng h/mL, PND 56 30 mg/kg: Both sexes: decreased food consumption, abnormal gait, decreased activity, prostration, and mydriasis Juvenile dogs: NOAEL: 1 mg/kg AUC0-24 ng h/mL (m/f): Week 1: 684/442, week 4: 420/300, week 20: 472/390, week 33: 448/334 LOAEL 5 mg/kg: Abnormal gait and prostration/loss of consciousness. After repeated dosing of 10 mg/kg in addition vocalization, trembling, and excessive scratching/chewing. Adult dogs: ≥1 mg/kg: Mild abnormal gait was observed. AUC0-24 ng h/mL (m/f): 203.28/175.95 (day 1), 293.43/234.85 (day 28), 281.41/264.35 (day 91)	Aside from higher exposure levels recorded in the juvenile rats on PND 7 (∼1.5- to 2-fold), exposures in both species were in the same range in juvenile and adult animals. No novel toxicity: In both species, major side effects consisted in behavioral signs attributed to exaggerated pharmacology. In the rat, secondary to behavioral changes, reduction of growth progression seems to occur at lower dose and exposure indicative of higher sensitivity. In juvenile rats, neuropathology evaluations of peripheral nervous system and CNS did not raise signs of neurotoxicity.
Epi60-18 years	Rat PND 28	Juvenile rats: (oral gavage) NOAEL: 30 mg/kg: Decreased activity, tremors, erected fur, partly closed eyes, shallow breathing, head shaking, abnormal gait and/or animal lying on side, salivation, incidental findings of minimal centrilobular hypertrophy in both males and females, and increased liver weight (reversible; no histopathology correlate). AUC0-tlast ng h/mL (m/f): PND 28: 5,364/12,658 ng h/mL, PND 111: 22,684/28,719 ng h/mL 10 mg/kg: Decreased activity and closed eyes, animal lying on side or head shake, salivation (before treatment), and minimal centrilobular hypertrophy in males Adult rats: (dietary admixture) NOAEL: 20 mg/kg: Decreased urine urea nitrogen and creatinine, decreased brain weight in males, increased heart weight in females, increased liver weight in females (no gross or microscopic correlates), and dose-related thyroid follicular cell hyperplasia/hypertrophy At 75 mg/kg: Similar findings plus mortality and loss of muscle tone in hindlimbs, hypoactivity, prostration, tremor, and salivation. TK data from adult rat studies are either missing or not comparable to juvenile data because of dietary admixture administration on the compound.	At the same dose levels, exposure in juvenile animals was similar in rats aged PND 21 (preliminary range finding study), 28, 35, or 41, but was about 1.5- to 3-fold lower compared to rats aged PND 111 (definitive study). The toxicity profile observed in the juvenile rats was similar as observed in adult animals, with the observations primarily limited to CNS effects and effects on the urinary bladder. Liver and thyroid changes were attributed to metabolic adaptation. Apparent increased severity of toxicity was observed in rats (preliminary range finding studies) given the drug from PND 21 compared to rats given the drug from PND 28 and older, based on the severity of the clinical signs inducing preterminally euthanasia occurring from 30 mg/kg in the younger rats and from 60 mg/kg in the older rats. No specific neuropathology evaluation was performed.
Nar16-18 years	PND 7	Intraperitoneal (IP) dosing was used to increase exposure in juvenile rats. Oral administration was used in adult rats. Juvenile rats: NOAEL: 15 mg/kg, IP (1,276.5 ng h/mL) with local effects at injection site. Increase in alveolar macrophages (minimal severity and reversible). LOAEL: 30 mg/kg, IP (2,762.5 ng h/mL) mortality, convulsive episodes were noted. Dose-dependent increase in alveolar macrophages. Adults rats: oral route NOAEL: 30 mg/kg (223 ng h/mL) by oral route, with limited liver weight increment (6%). LOAEL: doses >75 mg/kg (862 ng h/mL) mortality, “uncontrolled movements,” aggressive behavior, Straub tail, and excitation, followed by prostration/lassitude.	Higher exposure levels in juveniles due to IP versus oral administration in the adult animals. Exposure (AUC) decreased 3.5-fold from PND 7 to PND 34 and raised again at PND 70. No new toxicity. Mortality and behavioral signs observed at LOAEL doses in both juvenile (30 mg/kg) and adult animals (75 mg/kg). No definitive conclusion possible about sensitivity as no IP study was conducted in adult animals. CNS sensitivity in juvenile and adults seems similar on the basis of exposure comparison, but effects seem different.
Schi112-18 years	Rat PND 24	Juvenile rats: Treatment-related pharmacology effects (ptosis and underactivity) were observed at all doses (occasionally at 0.16 mg/kg). The signs became evident from the end of dosing and persisted until the end of the working day, but were not evident the following morning. Estrus cycle disruption was observed at the end of the study in all treated groups. NOAEL: 0.63 mg/kg in females due to possible treatment-related effects on learning and memory among females receiving 2.5 mg/kg/d observed during the treatment phase of the study. No similar tests were performed in adult rats. AUC0-24 on PND 31: 385/525 ng h/mL (m/f) NOAEL: 2.5 mg/kg in males AUC0-24 on PND 31: 1,543/2,056 ng h/mL (m/f) Adult rats: LOAEL: ≥0.63 mg/kg: Dose-related sedation, hyperreactivity to touch, ptosis, effects on body weight, clinical chemistry and hematology parameters, pseudopregnancy, mammary gland stimulation, prostate inflammation, and ↑ prolactin AUC0-24 (m/f): 0.63 mg/kg: 512/965 ng h/mL 2.5 mg/kg: 1,948/5,115 ng h/mL	About 2-fold lower exposure was observed in juvenile rats on PND 31 compared to adults given the same dose of 0.63 mg/kg. No exposure data are available at the end of the juvenile study to allow a good comparison with exposure during the 6-month adult study. LOAEL was defined in adult animals at the same dose as the NOAEL in juvenile rats (females, 0.63 mg/kg). But because of different study durations (7 weeks vs 6 months), and different end points assessing behavioral changes (eg, effects on learning and memory assessed only in juvenile rats), no clear comparison of toxicity profiles between juvenile and adult rats was possible. No neuropathology evaluation mentioned.
Schi210-18 years	Rat PND 14	Juvenile rats: ≥0.4 mg/kg: Dose-related behavioral (underactivity/immobility and ptosis from 15 minutes to 4 hours postdose), body weight, and food consumption changes were noted during the treatment period. Increased motor activity and associated better performance on rotarod were noted during the treatment-free period. AUC0-7: PND 14: 149 ng h/mL, PND 69: 70 ng h/mL ≥1.2 mg/kg: Effects on fertility 3.2 mg/kg: Death of one male AUC0-7: PND 14: 1,154 ng h/mL, PND 69: 583 ng h/mL Adult rats: Dose-related major effects on body weight, food consumption, hematology or serum chemistry parameters, or reproductive organs were noted from 0.5 to 5 mg/kg. Pharmacological response (reduced activity and ptosis) was similar than in juvenile rats.	At the same dose levels, AUC-based exposure in juvenile rats (PND 14) was approximately 2-fold higher compared to adult animals (PND 69). Novel effect observed in juvenile rats (increased motor activity during reversibility) possibly related to the pharmacological activity of Schi2 on animals with ongoing ontogeny of D2 receptors. Nevertheless, since similar neurobehavioral examinations were not performed in adult repeated-dose toxicity studies, it cannot be concluded with a sufficient degree of certainty that this effect is specific to juvenile animals. Pharmacological response (reduced activity and ptosis) was similar in juveniles as in adults. The juvenile rats do not seem more sensitive to the treatment than the adult rats. No treatment-related effect on absolute brain weight and rat brain histopathology.
Schi310-18 years	Rat PND 21Dog PND 56	Juvenile rats: At all doses: Increased motor activity and impaired learning and memory. The following changes were also noted: atrophy of the pituitary pars intermedia, adrenocortical hypertrophy, mammary gland lobular hyperplasia and increased secretion, cervical and vaginal mucosal mucification, uterine endometrial atrophy, and decreased total ovarian corpora lutea. ≥20 mg/kg: Ptosis and decreases in body weight, reduced food consumption, and delayed sexual maturation (f). 20 mg/kg was apparently a clearly active pharmacological dose. AUC: PND 28: 1,665 ng h/mL, PND 79: 9,830 ng h/mL (mean, m+f) 40 mg/kg: Moribundity or death of 8 males and 4 females and associated clinical signs. AUC: PND 28: 11,250 ng h/mL, PND 79: 24,300 ng h/mL (mean, m+f) Adult rats: In a 4-week oral toxicity study, atrophy of the pituitary pars intermedia and decreased pituitary weight (females) were recorded in rats given 60 or 100 mg/kg. In a 13-week oral toxicity study, the no-effect-level was 6 mg/kg in males and 2 mg/kg in females due to effects on reproductive organs observed. In a 26-week oral toxicity study, dose-related (10-60 mg/kg) exaggerated pharmacology effects and effects considered secondary to drug-related increases (females) or decreases (males) in serum prolactin levels. Pharmacologically mediated hypoactivity and ptosis after dosing followed by hyperactivity prior to dosing in both males and females were observed at 30 and 60 mg/kg. AUC: Study day 1: 13,555/34,874 ng h/mL (m/f), study day 177: 106,693/72,377 ng h/mL (m/f) Juvenile dogs: Pharmacologically mediated CNS-related clinical observations (ie, hypoactivity, tremors, ataxia, vomiting) and minimally to mildly reduced body weight gain were observed. Adult dogs: Oral toxicity data not available.	Exposure in juvenile rats on PND 28 was usually lower than on PND 79 (∼2- to 6-fold, 20 and 40 mg/kg), especially at higher dosages. Exposure increased also from study day 1 to 177 in the 6-month toxicity study in adult rats (∼2- to 8-fold, 60 mg/kg). In dogs, no data in adult dogs given the drug by oral route were available. No novel toxicity was observed in juvenile rats as compared to adults. A higher sensitivity of juvenile rats to pharmacology-related effects of the drug cannot be ruled out based on mortality data. Mortality was observed in juvenile animals given 40 mg/kg, while no mortalities were recorded in adult rats given 60 (similar exposure level than in juvenile at 40 mg/kg on PND 79) or 100 mg/kg, which seems to indicate a higher sensitivity of juvenile animals to Schi3. In rats, the comparison between juvenile and adult behavioral effects is hampered by the lack of detailed behavioral examination performed during the repeated-dose adult study. There was no evidence of neurotoxicity in a comprehensive microscopic evaluation of the brain (including morphometry) at doses up to 40 mg/kg/d in juvenile rats or 30 mg/kg in juvenile dogs.
Tou16-17 years	PND 21	Studies in juvenile or adult animals did not show direct organ toxicity but rather functional effects considered to result from an exaggerated pharmacodynamic effect of Tou1, particularly on the CNS and prolactin secretion. These effects were generally seen at lower exposure levels in juvenile rats (hypoprolactinemia, decreased body weight gains, and clinical pathology changes). Juvenile rats: NOAEL: 0.5 mg/kg with respect to prolactin effects (increase in ovary weight in females) Cmax: PND 21: 18.9 ng/mL, PND 69: 24.3 ng/mL AUC: PND 21: 138 ng h/mL, PND 69: 114 ng h/mL At higher doses, AUC increased 1.5- to 2-fold following treatment for 7 weeks. Adult rats: NOAEL: 4.0 mg/kg with respect to the minimal effects attributed to the pharmacological properties of the compound at this dose level. Cmax: 217 ng/mL (AUC data not available) Juvenile monkeys: NOAEL: 0.5 mg/kg with respect to pharmacology side effects of Tou1 including hypoprolactinemia level and the alteration of the ability to learn several complex behavioral tasks. AUC0-24: 568 ng h/mL Adult monkeys: NOAEL: 0.1 mg/kg with respect of severe agitation and hypotensive effect not observed during the juvenile study AUC: 139 ng·h/mL Exposure in adult rhesus monkeys (7 years) is similar as in juveniles at similar dose levels.	Similar exposure data (Cmax) were recorded at similar dose levels in juvenile and adult animals (based on Cmax in rats and AUC in the monkey). No new toxicity. In the rat, the pharmacological sensitivity appeared to be higher in juvenile animals. In both species, the behavioral effects detected during clinical observations or physical examinations were different in juvenile and adult animals, and a gender difference in the behavioral changes was detected. No toxicity observations were detected during brain neuropathology assessment in both species. In the rat, lower grade/incidence of prolactin-positive cells, both sexes, and decreased pituitary weigh were noted. These were considered secondary to the pharmacodynamic effect of Tou1.

Abbreviations: AUC, area under the curve; BID, twice daily; CNS, central nervous system; f, female; LOAEL, lowest-observed-adverse-effect level; H&E, hematoxylin and eosin; JAS, juvenile animal studies; m, male; Nar, narcolepsy; NOAEL, no-observed-adverse-effect-level; NOEL, no-observed-effect-level; PND, postnatal day; PO, orally; TK, toxicokinetic.

^aCompounds are anonymized in association with the intended indication: Adhd, attention-deficit hyperactivity disorder; Dep, depressive- and anxiety disorders; Epi, epilepsy and bipolar I disorders; Nar, narcolepsy; Schi, schizophrenia; Tou, De la Tourette and restless legs syndrome.

In dogs, exposure comparisons were possible for 5 compounds, as in one case no data in adult dogs were available. In 4 cases, the exposure was 2- to 3-fold higher in juvenile animals (starting age PNDs 4-70), whereas in one case the exposure was similar in juvenile and adult animals. In the one case of monkeys being used, there was similar exposure in juvenile (2-4 years of age) and adult animals.

Comparison of General Toxicity Between Juvenile and Adult Animals

Toxicity end points such as mortality, detailed clinical observations (see later), body weight and food consumption, clinical chemistry, and behavioral observations were included, as well as macroscopic examinations and common histopathology at necropsy. Reproductive end points (such as sexual development, hormone analysis, and reproductive assessments) were included in the majority of rat studies.

Supplementary end points such as bone measurements, ophthalmoscopy, and extended neuropathology were less frequently reported in the study reports. In Table 3 (fourth column), a conclusion on the comparison of exposure and toxicity in juvenile versus adult animals is presented for each individual compound. Differences between juvenile and adult rats in terms of increased severity of the effects were observed for 7 compounds (Tou1, Epi2, Epi3, Epi4, Epi5, Epi6, Schi3) and not for 8 other compounds (Nar1, Epi1, Adhd1, Adhd2, Schi1, Schi2, Dep1, Dep2).

In 8 cases, no novel toxicity was observed in juvenile animals as compared to adults. Effects seen in juvenile rats only, but secondary to pharmacological effects on growth, such as decreased food consumption and body weight (eg, delayed sexual maturation, femur length), were not considered as “new toxicity.” In 6 cases, novel CNS toxicities were observed, which are discussed later on.

In one case, Epi3, novel toxicity consisted of a bone effect, that is, reduced mineral bone content, area, and density of the femur and lumbar vertebrae in juvenile dogs. However, to our knowledge, bone mineral density had not been assessed in adult dogs, hence making a direct comparison impossible.

In all cases, the results of the JAS have been reported in the SmPC (with the exception of one case where the marketing authorization was suspended). The review of PDCO assessment reports did not identify any significant amendments of the clinical pediatric development caused by JAS outcomes for the products analyzed.

Screening of CNS Effects in Juvenile Animals

Examinations intended to detect CNS pharmacological or toxicological side effects were performed in most cases during the treatment period, usually also during the off-treatment “reversibility” period, and in one case, behavioral examinations were done during the reversibility period only.

The following qualitative observations were generally recorded in the rat studies:

Clinical observations intended to detect evidence of ill-health and/or mortality, cage observations performed twice or sometimes once daily.

Detailed clinical observations intended to detect pharmacological or toxicological side effects. Often, these observations were timed in relation to time of administration: prior to administration, just after administration, a couple of hours later on, and sometimes at the end of the afternoon. They generally consisted of cage observations, handling observations, and observations of reaction to manipulation.

Physical examinations performed routinely once a week.

Detailed clinical observations have also been recorded in all the nonrodent studies. In addition, in some of the nonrodent studies, operant behavior was assessed.

Behavioral examinations in the rat studies, which were usually assessed a few weeks after start of treatment (sometimes just before daily dosing), generally consisted of:

Open field observation/functional observatory battery (FOB), or modified Irwin

Auditory (or acoustic) startle reflex testing

Learning and memory testing: Water maze tests were used for these purposes. ⁹ These tests were conducted at ages >PND 50, usually once during treatment, and once during off-treatment. Morris ¹⁰ or Cincinnati ¹¹ water maze tests were most commonly used (Tou1, Adhd1, Adhd2, Epi1, Epi5, Epi6, Schi2, Schi2, Dep1, and Dep2), while in 2 cases (Nar1, Schi3), the type of water maze was not specified. In 2 cases, other water mazes were used, including Biel ¹² or multiple T-maze ¹³ (Epi2, Epi4).

Motor activity monitoring

In juvenile rats, various extended neuropathology evaluations (eg, colorations intended to detect pharmacologic or neurotoxic effects on neurons, apoptosis, myelinization) of peripheral nervous system and CNS were performed in 8 of 15 cases.

Effects on CNS Measures Monitored During JAS

An overview of the CNS end points included in each study and their respective outcomes is presented in Tables 4 (rats) and 5 (nonrodents). The effects of CNS detected through detailed clinical observations were noted for nearly all compounds during the treatment phase (eg, salivation, increased/decreased activity, stereotypic behavior, convulsions, abnormal gait, ptosis, tenseness, vocalization, aggressivity) but not during the off-treatment (reversibility) phase.

Table 4.

Effects on CNS Measures in Rodents.^a

Compoundb	Observations		FOB/Irwin		Auditory startle		Water maze		Motor activity
	T	R	T	R	T	R	T	R	T	R
Adhd1 13-W starting on PND 7 + 4-week treatment-free period	Clin obs: DailyDetailed timed obs: PND 92Phys ex: 1/W	Clin obs: DailyDetailed timed obs: PND 99	ND	ND	ND	ND	ND	ND	ND	ND
	HD (f), during study: Occasional vocalizationHD, from PND 80: No movement, decreased activity, and unsteady/abnormal gait, fixed gazeMD and HD, from PND 80: Increased agitation, tenseness, vocalization, and/or aggressiveness at dosing or handling	No effects	ND	ND	ND	ND	ND	ND	ND	ND
Adhd1 7.5-W starting on PND 7, no treatment-free period	Clin obs: DailyDetailed timed obs: DailyPhys ex: 1/WPND 20: Visual function/placement	ND	ND	ND	PND 20	ND	Morris water maze	ND	PNDs 44-52	ND
	No effects	ND	ND	ND	No effects	ND	No effects	ND	MD (m): Higher rearing activity, higher maximum time on rotarodHD (f): Higher activity, higher maximum time on rotarod	ND
Adhd2 8-week starting on PND 7 + 4-week treatment-free period	Clin obs: 2/DDetailed obs: 2/D until weaning, then 1/WCage obs: 2 hours postdose (after weaning)		PND 22/23 and PND 59/60 prior dosing	PND 88/89	PND 62/63 prior dosing	PND 90/91	Cincinnati water mazePND 52 and PND 61 prior dosing	PNDs 78-84	PND 22/23 and PND 59/60Prior dosing	PND 88/89
	All doses: Increased activityHD: Stereotypic behavior	No effects	No effects	No effects	HD males: Lower average startle	No effects	No effects	No effects	HD (m) and all doses (f): Lower activity counts	No effects
Dep1 10-week starting on PND 21 + 4-week treatment-free periodTwice-daily oral gavage	Clin obs: 2/DTimed detailed obs: At least 2/WPhys obs: 1/W	Clin obs: 2/D	ND	ND	PND 59 (2 hours after first dose)	PND 114	Morris water mazePND 61, (2 hours after the first dose)	ND	PND 58 (2 hours after first dose)	PND 113 males
	All doses, dose related: Chin rubbing, and occasionally paddling of the forelimbs and/or salivation	No effects	ND	ND	HD: Low amplitude increases (both sexes)	No effects	No effects	ND	All doses, not dose relatedSlightly higher scores, males, attributed to atypical control values	No effects
Dep2 10-week starting on PND 7 + 4 to 7-week treatment-free period	Clin obs 2/DCage 3 hours postdose: dailyDetailed obs: prior dosing: daily	Clin obs: 2/DDetailed obs: 2/W	ND	ND	PND 28	ND	ND	PNWs 12-14: Cincinnati water mazePNW 12: Passive avoidance test	ND	PND 91
	MD and HD, dose relatedDecreased activity and respiratory rate up to PND 14	No effects	ND	ND	No effects	ND	ND	No effects	ND	No effects
Epi1 10-week starting on PND 7 + 10-week treatment-free period	Clin obs: 2/DDetailed clin obs: Daily	Clin obs: 2/DDetailed clin obs: Daily	Functional development before PND 21FOB, weeks 4, 7, and 10	Week 12FOB	Weeks 4, 7, and 10	Week 12	Cincinnati water mazeWeeks 10/11	Cincinnati water mazeWeeks 12/13	Weeks 4, 7, and 10	Week 12
	HD: Decreased activity, mainly preweaning	No effects	No effects	No effects	HD: W4, femalesIncreases in maximum and average startle	No effects	No effects attributed to treatment	No effects	No effects	No effects
Epi2 5-week starting on PND 12 + 2-week treatment-free period	Clin obs: 2/DTimed observations (recorded 0.5-3 hours postdose)	Clin obs: 2/D	PND 46 (before dosing)Grip strength, pupillary reflex, visual stimulus	PND 66Grip strength, pupillary reflex, visual stimulus	PND 46 (before dosing)	PND 66	Water multiple T-mazePND 35 to PND 37 and PND 48 to PND 51 (before dosing)	PND 66 and PND 80	PND 46 (before dosing)	PND 66
	HD (after dosing): Tremor, hypotonia, abnormal gait.MD (after dosing): Hypotonia	No effects	No effects	No effects	No effects	No effects	Impaired learning: HD both sexesMemory, errors: Not consistent between doses and sexes	HD, both sexes: Better water maze test performance	No effects	No effects
Epi4 9-week starting on PND 4 +4-week treatment-free periodBID administration	Clin obs: 2/DTimed obs: Daily up to euthanasia	Clin obs: 2/D	PND 22 (4 hours after first daily subdose)	PND 77	PND 23 (4 hours after first daily subdose)	PND 78	Biel maze swimming testStarting PND 24, PND 22 (4 hours after first daily subdose)	Starting PND 78	Locomotor activity, PND 22, 4 hours postdose	Locomotor activity, PND 77−
	No effects	No effects	No effects	No effects	No effects	HD (m, f): Increased magnitude of the response	No effects	No effects	HD (m): Decreased mean total and ambulatory motor activity counts (0-15 minutes only)	No effects
Epi5 12-week starting on PND 7 + 4-week treatment-free period	Clin obs: 2/DTimed clin obs	Clin obs: 2/D	Qualitative and quantitative, weeks 5 and 11	Week 16	Weeks 5 and 11	Week 16	Cincinnati water mazeWeeks 10 and 12	Cincinnati water mazeWeek 16/17	Weeks 5 and 11	Week 16
	MD and HD groups, postdosing: Decreased activity, uncoordination, excessive grooming/licking/scratching and ptosis	No effects	MD and HD: Decreased grip strength (attributed to small size)	No effects	No effects	No effects	No effects	No effects	No effects	No effects
Epi6 12-week starting on PND 28 + 4-week treatment-free period	Clin obs: 2/DDaily timed obsDetailed obs: 1/W	Clin obs: 2/DDetailed obs: 1/W	Qualitative obs: Prior treatment, PNWs 6 and 15	PNW 19	Prior treatment, PNWs 6 and 15	PNW 19	Cincinnati water maze: Prior treatment, PNWs 13 and 14	PNWs 18 and 19	Prior treatment, PNWs 6 and 15	PNW 19
	HD: Transient (PNDs 28-31), decreasing with repeated administration:Decreased activity, tremor, head shaking, abnormal gait, convulsions	No effects	No effects	No effects	No effects	No effects	No effects	No effects	No effects	No effects
Nar1 9-week starting on PND 7 + 3-week treatment-free periodIntraperitoneal route (BID for control and high-dose groups)	Clin obs: 2/DTimed clin obs: DailyPhys ex: 1/W	Clin obs: 2/D	At least 5 hours after first daily treatmentPND 8 righting reflexPND 17: Grip strength, pup functional developmentPND 21: Pupillary and auditory reflexW7 of age: Open field test (exploration)	ND	ND	ND	W5 and W6 of age, at least 5 hours after first daily treatmentType of Water maze, not specified	ND	W7 of age, at least 5 hours after first daily treatment	ND
	HD (once or BID):Immediate postdosing convulsive episodes	No effects	No effects	ND	ND	ND	No effects	ND	No effects	ND
Schi1 7-week starting on PND 24 + 6 to 8-week treatment-free period	Daily obs: 2/DDetailed timed obs: Daily for 1 week, then 2/W	Daily obs 2/D	PND 63/64 prior dosing	PND 87/88	ND	ND	Morris water mazePNDs 67-69 prior dosing	Morris water mazePNDs 92-96	PNDs 65-66 prior dosing	PND 89/91
	All doses, dose relatedPtosis, underactivity	No effects	No effects	No effects	ND	ND	HD (f): Longer trial times, high failures rate = lower improvement of performance during the test	No effects	No effects	No effects
Schi2 8-week starting on PND 14 + 6-week (f) or 8-week (m) treatment-free periodSC administration	Clin obs: 2/DTimed obs: Daily for 3 weeks, then 2/WPhys ex: 1/W after weaning	Clin obs: 2/D	ND	Locomotor coordination (rotarod) PND 76/78	ND	PND 79/80	ND	Morris water mazeStarting PND 82/84	ND	Locomotor and rearing activityPND 74/75, PND 81, and PND 99
	All doses, dose relatedImmobility, ptosis, reduced activity	No effects	ND	HD: Better performance	ND	No effects	ND	No effects	ND	All doses, dose related: High motor activityD30 (PND 99) of recovery: Signs of recovery in males, but not in females
Schi3 2-month starting on PND 21 + 4 to 10-week treatment-free period	Clin obs: 2/DDetailed timed obs: Daily	Clin obs: 2/DOnce a day detailed observations	FOB: PNDs 40-46	PNDs 108-109	PNDs 47-52	PNDs 11-112	Water mazeType not specifiedPNDs 68-72	Water mazePNDs 113-116	PNDs 56-60	PNDs 116-118
	MD and HD: PtosisHD: Splayed hindlimbs, decreased activity	No effects	No effects	No effects	No effects	No effects	HD (m) and all doses (f): Impaired learning and memory deficit	No effects	All doses, nondose relatedIncreased motor activity	HD (f): Increase persisted in females
Tou1 7-week starting on PND 21 + 4-week treatment-free periodOral gavage	Clin obs: 2/DDetailed timed obs: At least 2/W up to W7Phys ex: 1/W	Clin obs: 2/D	Hand/Arena obs: W6, prior Treatment	Hand/Arena obs: W3/4 of recovery	W7, prior treatment	W3 of recovery	Morris Water mazeW7, prior treatment	W4 of recovery	W6, prior treatment	W3 and W10/11 of recovery
	Dose response, all doses: Underreactive (f) and overreactive (m/f) behavior and partially closed eyelids	No effects	No effects	No effects	MD and HD: Low-peak amplitude and habituation (m)Quicker mean response (f)	No effects	MD and HD: Decreased performance and failed water maze trials	No effects	HD (f): High ambulatory and rearing activity	HD (m): Low ambulatory and rearing activity persisting up to end recovery

Abbreviations: BID, twice daily; Clin obs, clinical observations; +, CNS treatment effects; D, study day; f, female; FOB, functional observational battery; HD, high dose; IP, intraperitoneally; m, male; MD, mid dose; ND, no data/not done; −, no difference from controls; Phys ex, physical examinations; PND, postnatal day; PNW, postnatal week; SC, subcutaneously; W, study week.

^aFor Epi3, no rodent studies were available.

^bCompounds are anonymized in association with the intended indication: Adhd, attention-deficit hyperactivity disorder; Dep, depressive and anxiety disorders; Epi, epilepsy and bipolar I disorders; Nar, narcolepsy; Schi, schizophrenia; Tou, De la Tourette and restless legs syndrome.

Table 5.

Effects on CNS Measures in Nonrodents.^a

Compound	Detailed clinical observations	Operant test battery	Species
Adhd2	+	−	Dog
Epi1	−	−	Dog
Epi2	+	ND	Dog
Epi3	+	ND	Dog
Epi4	+	ND	Dog
Epi5	+	−	Dog
Schi3	+	ND	Dog
Tou1	+	+	Monkey

Abbreviations: Adhd, attention-deficit hyperactivity disorder; CNS, central nervous system; Dep, depressive and anxiety disorders; Epi, epilepsy and bipolar I disorders; ND, no data/not done; +, CNS treatment effects; −, no difference from controls; Nar, narcolepsy; Schi, schizophrenia.

^aFor Adhd1, Dep1, Dep2, Epi6, Nar1, Schi1, and Schi2, no nonrodent studies were available.

In 7 of the 8 nonrodent JAS, effects were observed through detailed clinical observations. For operant behavior, only effects were observed in the monkey study with Tou1. The operant test battery aimed at assessing learning, short-term memory, color discrimination, and motivation. Other compounds, such as Adhd2, Epi1, and Epi5, did not induce any effect in these end points.

During FOB/Irwin testing, no effects were noted for any compound during the treatment period. An effect on rotarod performance, most probably related to an increased motor activity, was recorded for Schi2 during the off-treatment period (not tested during the treatment phase).

Acoustic startle reflex (Table 4, third column) was affected by Tou1, Adhd2, and Epi1 as well as by Dep1. Effects were mainly seen at high dosages. Statistical difference from controls was seen in startle reflex data for Epi4 during the off-treatment period, but not during the treatment period.

Effects on water maze were reported for Tou1, Epi2, Schi1 (only females), and Schi3. For Epi2, learning alterations and memory deficit (eg, worse water maze performance) were noted during the treatment period. The 4 studies with water maze findings also included a water maze assessment during the off-treatment period, but none revealed adverse alterations during the off-treatment period.

Motor activity was affected by Tou1 (increased), Adhd1 (increased), Adhd2 (decreased), Epi4 (decreased), Schi3 (increased), Dep1 (increased) during the treatment phase while not being an end point for Schi2, and Dep2 during the treatment phase. Motor activity differences from control were still seen during the off-treatment phase with Tou1 (decreased motor activity, possibly a rebound effect) and Schi3 (increase persisted in females). Motor activity was measured during the off-treatment period only and found to be different from control data for Schi2 (increased signs of recovery in males, but not in females, also observed in adult study). Extended neuropathological histopathology investigations did not show signs of neurotoxicity for any of the compounds.

Comparison of the behavioral profiles between juvenile and adult animals is impossible as behavioral end points used in JAS were not addressed during the adult toxicity studies. Nevertheless, detailed clinical observations were recorded for different ages of animals during the JAS, and the evolution of these observations from the youngest age to the young adult rats was informative in this respect.

Age-related novel CNS effects were observed for 6 compounds: Tou1, Epi4, Adhd1, Dep2, Schi1, and Schi2. For example, in male rats of the rat JAS for Tou1 underactivity predominated, while in females initially tending to be underactive, overactive behavior increased as treatment progressed. Increased motor activity was seen afterward, during the off-treatment period, possibly a rebound effect. This was not described for adult animals. In the monkey study with Tou1, different behavioral effects were observed in juvenile animals compared to adult monkeys, as evidenced by toxicity (severe agitation resulting in wounds and bruises at the 2 higher doses) occurring in adult animals only. In juvenile monkeys, alteration of the ability to learn complex behavioral tasks was noted. These tests were not performed in adult monkeys. Similarly, the rat JAS for Adhd1 revealed behavioral effects at the end of the dosing period (increased agitation, tenseness, vocalization, aggressiveness, followed by decreased activity/no movement/fixed gaze starting on PND 80), whereas no similar clinical CNS signs were reported either in younger rats or in the adult rat study.

Juvenile animals exposed to Tou1, Epi3, and Epi5 appeared more sensitive to the pharmacological activity or CNS clinical signs. In the case of Epi2, Epi4, and Schi3, mortality, possibly linked to exaggerated pharmacology, was observed in young animals at an exposure that was not lethal in their adult counterparts.

Regarding Epi 6, the pivotal rat JAS (PND 28) showed a similar toxicity profile as observed in adult animals. However, in the dose-range finding study performed in younger animals (PND 21), notwithstanding similar exposures, more severe clinical signs inducing preterminal euthanasia were reported in these animals.

Discussion

The present article summarizes the review of the juvenile safety studies conducted with 15 CNS-targeted compounds. The selection of compounds was conditioned by the availability of adequate data in the EMA archives. We have chosen to focus on compounds with a direct CNS target and to leave out compounds that only have an indirect effect on CNS conditions (eg, compounds with a target in the immune system for the treatment of multiple sclerosis). The studies performed for the 15 compounds reveal a number of issues with regard to the study design.

Species Selection

In the case of antiepileptic drugs, the choice of 2 species for 4 of the 6 compounds is not fully understood. In general, neither the choice of the species nor the number of species used for toxicity testing in juvenile animals was justified in the available documents. The choice of a second species might have been related to the outcome of some rodent studies in previous programs for pediatric antiepileptics, that is, with an observed decrease in brain weight leading to further questions regarding the relevance of this effect (Epi1).

Regulatory guidelines indicate that usually one (rodent) species would be sufficient, and the rat is seen as the default species. Selection of an adequate animal species, no matter whether rodent or nonrodent, should be scientifically justified whenever possible. This is especially important considering the limitations of JAS in nonrodents for CNS drugs: difficulty to treat very young animals up to adulthood, difficult access to appropriate tests, and ethical and practical challenges of obtaining and treating preweaning nonhuman primates.

Starting Age

There was notable variety of the age of juvenile rats and dogs at study start, which did not always correspond to the youngest age of the intended patient population. Of particular interest was Epi6, where the single pivotal JAS was conducted in 28-day-old rats, which is too old to represent the stage of CNS development in the intended population (preterm neonates) as a 28-day-old rat would rather correspond to a 4-year-old in terms of brain development. ¹⁴ In addition, apparent increased severity of toxicity was observed in rats given the drug from PND 21 compared to rats that were given the drug from PND 28 and older, indicating higher sensitivity in younger rats, based on the severity of the clinical signs leading to preterminal euthanasia occurring at lower doses in the youngest rats. Consequently, the critical window of vulnerability of the developing brain and any deleterious effects of Epi6 associated therewith could have been missed. Therefore, it remains uncertain to what extent the observation of a similar toxicity profile in the pivotal JAS compared to adult animals can be extrapolated to the youngest children.

On the contrary, PND 7 as starting age for the juvenile rat studies with Adhd1, Adhd2, Dep2, and Nar1 with a target population from 6 or 7 years of age onward is considered too young. Before PND 10, the rat is comparable to a human fetus or a preterm neonate. Therefore, safety signals in such young rats may be irrelevant for humans based on differences in developmental stages. ¹⁵ In the case of Adhd1, the clinical relevance of the novel behavioral effects at the end of the juvenile rat study (increased agitation, tenseness, vocalization, and aggressiveness, followed by decreased activity/no movement/fixed gaze) is questionable in view of the immature rat brain at the start of the study compared to the target age in children. However, these findings might be related to the neuropharmacological properties of Adhd1, which may potentially affect neurodevelopmental processes in early phases of brain development, leading to changes in neuropharmacological response and behavioral outcome at later stages in life. Hence, these findings may also reflect a particularly sensitive period in these young adult animals and be relevant for adolescent patients.

In very young rats, excessive systemic and brain exposure related to physiological and metabolic immaturity may induce severe toxicities of little clinical relevance. For Dep2, the very early dosing could indeed be an explanation for the sharp increase in systemic exposure at the study start. Dosing on PND 7 led to 37-fold higher AUC values compared to PND 76. At the lowest dose, changes in body weight, physical development, and pituitary weight were observed in males, and a no-observed-adverse-effect-level (NOAEL) could not be determined. The effects did not reverse during the recovery period and are novel toxicities compared to previously available nonclinical data for this compound. However, at the lowest dose in the JAS, at PND 7, the exposure (in terms of AUC) was still twice the exposure at the NOAEL of the comparative adult rat study, and more than 700 times the AUC at the clinical dose (5 mg/d in 6-17-year-old patients), putting the relevance of these novel findings into question.

In conclusion, whereas in regulatory guidelines, the starting point is that the age range of juvenile animals should cover the window of brain development corresponding to the children’s treatment period, this principle was not respected in several of the reviewed JAS and as a result, the clinical relevance of these study results is questionable. Animal age at study start should be scientifically justified to improve the clinical relevance of the studies. The choice of 2 species and the variety in starting age might be related to the time period in which the studies were conducted, before US Food and Drug Administration (FDA) and EU guidelines ^2,3 had been published and before the EU PIP procedure came into force in 2007.

Study Duration

In spite of the variability in the starting age of JAS mentioned above, the study duration was more harmonized as most studies covered the full developmental trajectory of the brain with a majority of the rat studies including dosing up to at least PND 70. Short-term studies focusing on juveniles as compared to adult periods of sensitivity to the pharmacological or toxicological effects are not very common. This may be due to the assumption that CNS behavioral effects could be delayed or long lasting even after cessation of compound administration throughout brain development up to adulthood.

In one case, severe CNS effects were seen in rats aged PND 80 and older. These effects were not detected in younger or adult rats pointing out to the need to cover brain development up to adulthood.

Number of Animals Used

The number of animals used in this type of safety studies is an important concern, in relation to the information gained. In this analysis, the number of juvenile animals on study is quite variable and in some cases very high. The highest numbers were observed in complex studies which covered multiple end points requiring additional cohorts, particularly pup’s reproductive capacity in rat JAS.

In an effort to reduce the number of animals needed, study designs should be streamlined and investigated end points scientifically justified and reduced to the most relevant ones. In addition, when studies are started with rats preweaning, additional pretest animals are needed in order to avoid confounding factors (eg, genetics, group-size during fostering) between groups susceptible to interfere with study results. Usually, groups are built from as many litters as are present in the study (ICH, S5 ¹⁶ ). It has, however, to be established whether these principles should be applied stringently or if a more flexible approach may be adopted in the interest of reducing the number of animals used for these studies.

Use of Excipients

Usually, excipients used in JAS are similar to those used in the adult formulations. Nevertheless, they may have a different safety profile in very young animals. The use of propylene glycol as vehicle for Epi6 led to excessive mortality in young mice. These data are in-line with the results of a juvenile (PND 7) mouse study, ¹⁷ which showed that propylene glycol produces ethanol-like apoptotic neurodegeneration in the developing mouse CNS starting at doses of 2 mL/kg. At 1 mL/kg, no apoptotic effects were observed. At 10 mL/kg, it induced mortality (unpublished results). Although excipient toxicities may seriously hamper the interpretation of the effects caused by the active ingredient, using a clinical formulation for the JAS can be useful to assess the safety of the clinical formulation and the need for a specific pediatric formulation.

Toxicokinetics

Toxicokinetic data are important to understand whether the test results occurred in a similar exposure range as the intended patient exposure. Moreover, they are critical to assess the impact of changes in systemic exposure across age ranges on the observed toxicities. Therefore, it is desirable to measure TK shortly after the study start and at the end of the study. An additional sampling around the time of weaning could also be useful in some cases. It should be kept in mind, however, that age-related changes in pharmacokinetics cannot be directly extrapolated from animals to humans. Estimation of kinetic properties in humans rather stems from knowledge of developmental changes of enzymes and organs influencing pharmacokinetics in humans, as well as modeling. The need to evaluate brain exposure should be properly addressed taking into account potential differences in brain penetration between species, although it has to be admitted that for none of the compounds included brain exposure was determined.

CNS Effects in Juvenile Animals

Effects have been observed during detailed clinical observations, but generally no effects were detected during specific behavioral tests, such as the FOB. The timing of the detailed observations in relation to dosing (before and several times following dosing) might explain why these effects were not observed during the other behavioral examinations (usually performed once/twice at the end of the study; often just before dosing and during the off-treatment period). This apparent discrepancy may also be due to the kind of end points evaluated. During detailed clinical observations, a broad spectrum of mostly qualitative end points was evaluated. During behavioral examinations, specific end points were measured. As effects were rarely seen just before dosing (unless at high doses) or during the off-treatment period, results indicate that most effects are related to drug exposure and are not long lasting.

Effects on acoustic startle and water maze tests were only seen with a low number of compounds, mostly at mid and/or high doses. These effects were often not consistent between doses and sexes. In one case, a change in auditory startle response was detected only during the off-treatment period. In another case, learning deficits were detected in groups previously treated with the high dose, whereas memory task errors were noted at all doses, which were not, however, consistent regarding dose dependency or between sexes. This may indicate a low sensitivity of these tests. As these tests were not performed during adult toxicity studies, it is also not possible to determine whether these effects are specific to juvenile animals.

Effects on motor activity were seen in 6 of 12 compounds during the treatment period and in 3 cases during the off-treatment period. The effects observed during the off-treatment period are not easy to interpret. For Tou1, a decrease in motor activity might have been a rebound effect caused by the stimulation of motor activity during treatment. In the case of Schi2, off-treatment effects were observed on rotarod performance and on motor activity (high locomotor and rearing activity), at all doses and dose related. These effects tended to decrease in male but persisted in females until the end of the recovery period. As no tests were carried out during the treatment period, it is again difficult to interpret these effects. For Schi3, nondose related but statistically significant increases in motor activity were observed both in males and females at all doses tested during the treatment period. In males, the effect on motor activity resolved during the off-treatment period while in females the effect on motor activity persisted during the drug-free recovery period at the highest dose tested. Long-term effects of antipsychotics, such are tardive dyskinesia, are also well-known in humans, but their relevance for children will have to be confirmed by clinical data.

Impact of Ontogeny of Target Systems

Various receptors are being involved with these 15 compounds, including dopamine receptors (covering 5 compounds: Adhd2, Tou1, Schi1, Schi2, and Schi3), α2-adrenergic receptors (Adhd1), sodium channels (Epi1, Epi2, and Epi3), potassium channels (Epi6), AMPA glutamate receptor (Epi5), and histamine H3 receptors (Nar1). Other compounds have a less clearly defined receptor interaction.

There is a lot of published literature regarding the ontogeny of dopamine receptors in rats. D1 and D2 receptors follow similar developmental patterns, showing a peak between PND 28 and PND 42 in the caudate and putamen, and decreasing thereafter. ^18,19 However, the picture is more complicated as dopamine also occurs in the ventral striatum. The pattern of maturation of the dopaminergic innervation is, however, less clear in the frontal cortex. ²⁰ For other receptors and channels, identified literature ^{21 -23} was not very helpful to understand the relevance of the observations.

Overall, in the absence of a discussion by the sponsors on the findings in relation to the ontogeny of the receptors or the relationship between the observations in juveniles as compared to their effects in adults, an in-depth elaboration on the behavioral findings observed with the various dopaminergic (agonists and antagonists) or other compounds was not considered feasible within the remits of this study.

Lessons Learned With Regard to Screening of CNS Effects in Juvenile Animals

Detailed Clinical Observations

Behavioral observations, although only qualitative, are important because they may help to detect severe abnormalities, such as convulsions, aggressiveness, or catalepsy. As they are performed at several time points, including just before daily administration of the drug and during the off-treatment period, they allow to differentiate high-dose or secondary short-term pharmacological effects from long-term or delayed pharmacological effects and/or neurotoxicity. In most cases, these observations can also be bridged to the adult animal data.

To reach these objectives, it would suffice to perform detailed clinical observations at the time of the highest exposure (peak exposure) and shortly before the next dosing (trough level). The severity of the effects should be related to exposure data.

Behavioral Examinations (Rat Studies)

Quantitative instrumental behavioral examinations whenever tested (not all were evaluated for each drug) showed lower incidences of treatment effects (as compared to the detailed clinical observations), and as these tests were not performed during adult toxicity studies, it is difficult to determine whether the observed effects were specific to juvenile animals.

During FOB testing, treatment effects were detected in only 1 case of 11 studies. Compared to the high rate of positive effects detected during detailed clinical examinations, this seems to indicate a low sensitivity of the FOB test and endorses a recent similar observation for pesticides ²⁴ . Considering that FOB is not quick or simple, the need to include them in JAS is thus questioned.

Treatment effects on acoustic startle and water maze tests were also seen in few cases, mostly at mid and/or high doses, often not consistently between doses and sexes. Of note, different maze tests were performed and their choice was not justified. They are all aimed at detection of learning and memory deficits, while testing for the brain networks that mediate higher cognitive functions need to include assessments of working memory, attention, long-term memory, and executive functions such as cognitive flexibility, not all being detected by one test. ²⁴ If these tests are thought to be needed to assess the hazard for impacting cognitive function in children and adolescents, the most relevant test should be selected and justified.

During (loco)motor activity testing, treatment effects were more frequently detected, including during the off-treatment period. As hyperactivity and hypoactivity are easy to detect, it may sometimes reflect rebound effects and—as was shown in 2 studies—may also reflect long-term effects during the off-treatment period, it may be wise to measure motor activity in complement to detailed clinical observations.

The need for more detailed behavioral assessment should, therefore, be decided on a case-by-case basis, based on specific concerns, in order to avoid overloading the studies. On the other hand, more detailed evaluation during the recovery phase may be needed if CNS effects are seen during treatment or are suspected to bear potential delayed effects, in order to assess reversibility versus long-term/delayed toxicity.

These data are consistent with the results of an Environmental Protection Agency review ²⁴ of 69 neurodevelopmental toxicity studies on pesticides, mostly neurotoxicants. Across the 69 studies, 2 types of neurobehavioral tests showed multiple positive results, locomotor activity and auditory startle response, whereas 2 other behavioral tests FOB and Learning and Memory (L&M tests) underperformed. Although Vorhees et al recommend dropping the FOB, they also suggest possible explanations for the apparent low sensitivity of L&M tests and propose reasons why they should still be performed, and how the selection of relevant neurocognitive tests could be improved taking into account the different cognitive functions covered by the Morris, Cincinnati, Radial Water Maze tests, and the Conditioned Fear test. Moreover, they suggest running these tests in young adult animals, as this test age is expected to result in better performance. The authors insist on the use of validated, standardized, automated, and scientifically chosen tests.

The incidence of positive findings in our project is also roughly consistent with recently published data from a similar research project conducted by the FDA Divisions on Psychiatry and Neurology Drugs, ²⁵ particularly the low incidence of positive findings for the FOB. Effects were observed during off-treatment for 5 of 14 drugs, that is, in 36% of the studies, compared to the 54% reported by FDA Divisions just mentioned.

The slightly lower scores of positive findings for some of the behavioral tests observed in our project are difficult to interpret. The Center for Drug Evaluation and Research project analyzed a number of studies twice as high as in the present study, that is, a different data set, although a partial overlap of a number of compounds may be possible. In addition, the timing of the tests has not been discussed; therefore, it is not known whether the observations were always done just prior to the daily treatment (trough exposure to the drug) or at the C_max (highest exposure to the drug).

Conclusions

This limited data set does not allow us to make clear recommendations regarding which concerns should trigger JAS for CNS compounds. It is important to have a strong scientific justification for each study, and dialogue with regulatory authorities before the conduct of this type of studies is recommended.

New toxicities or higher sensitivity to the treatment were detected in JAS compared to adult studies, but it was not possible to relate these effects to clinical situations. Nevertheless, multiple pediatric epidemiological studies showing convergence are usually needed to properly assess neurotoxicity in the clinic. The evaluation of CNS in daily practice is often difficult, as the methodology required for systematic results is difficult to implement in children with a high variety of confounders. From this point of view, it is important to study the behavioral effects in animals as it might be the only way to learn whether these compounds have adverse effects on daily functioning.

The evaluation of the designs and results of these studies highlighted which end points are more susceptible to yielding relevant data, particularly to detecting pharmacological and/or neurotoxic effects. Clearly, detailed clinical observations at specific time points in relation to time of administration are the most informative. Behavioral examinations, on the other hand, should be done only if scientifically justified and using well-standardized procedures.

Finally, the evaluation of the study designs stressed the high variability of these; sometimes not supporting the patient population to be treated (with respect to starting age and from an organ/systems maturation point of view), overly complicated, and therefore susceptible to induce irrelevant effects and/or to confound the readout, and using very high number of animals. It is important, based up experience, to streamline these study designs in order to obtain the optimal level of information. The companies responsible for development of the compounds reviewed in this article kindly agreed to publish the information on their products in its present format.