Clinical trial NCT02735473
Conditions: Fetal Alcohol Spectrum Disorders
Sponsor: University of Minnesota
Start date: 2016-06-01; Completion date: 2021-09-10
Enrollment: 46 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a randomized, double-blind, placebo-controlled trial of choline supplementation in children with Fetal Alcohol Spectrum Disorders (FASD). The primary outcome measures are cognitive measures.

Detailed description:
This project is the third in a series of randomized, double-blind placebo-controlled trials of choline bitartrate in children ages 2-5 who have been diagnosed with Fetal Alcohol Spectrum Disorders (FASD). Pre-clinical data suggests that choline may attenuate the cognitive deficits caused by prenatal alcohol exposure (especially memory deficits). This study will evaluate the effects of daily choline supplementation vs. placebo for 9 months. Outcome measures include an elicited imitation memory paradigm, global cognitive functioning, and aspects of executive functioning. The study will also continue to collect safety and tolerability data.

Arms: arm 1: Powdered drink mix containing choline bitartrate 19 mg. per kg. arm 2: Powdered drink mix containing matching placebo
Interventions: intervention 1: Choline bitartrate intervention 2: Placebo
Intervention descriptions: intervention 1: None intervention 2: None

Protocol documents:
Prot_SAP_001.pdf: 
University of Minnesota 
Protocol Design Template © 2007 
 
 
Choline Supplementation as a Neurodevelopmental Intervention 
in Fetal Alcohol Spectrum Disorders 
 
 
Regulatory Sponsor: 
Jeffrey R. Wozniak, Ph.D. 
Department of Psychiatry 
University of Minnesota 
(612) 273-9741 
Funding Sponsor: 
National Institute on Alcohol Abuse and Alcoholism 
Study Product: 
Choline 
Protocol Number: 
1R01AA024123 
IND/IDE Number: 
107085 
 
 
 
 
 
 
 
Date: 
7/29/2015 
Amended: 
1/08/2020 
Administrative 
Change: 
 
 
CONFIDENTIAL 
 
This document is confidential and the property of the University of Minnesota. 
No part of it may be transmitted, reproduced, published, or used by other persons 
without prior written authorization from the study sponsor. 


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Table of Contents 
STUDY SUMMARY ...................................................................................................................................... 1 
1 
INTRODUCTION ..............................................................................................................................................2 
1.1 
BACKGROUND ........................................................................................................................................................... 2 
1.2 
INVESTIGATIONAL AGENT ........................................................................................................................................ 3 
1.3 
PRECLINICAL DATA................................................................................................................................................... 4 
1.4 
CLINICAL DATA TO DATE ......................................................................................................................................... 7 
1.5 
DOSE RATIONALE AND RISK/BENEFITS ................................................................................................................ 8 
2 
STUDY OBJECTIVES ....................................................................................................................... 10 
3 
STUDY DESIGN ................................................................................................................................. 10 
3.1 
GENERAL DESIGN ................................................................................................................................................... 10 
3.2 
SECONDARY STUDY ENDPOINTS ......................................................................................................................... 11 
3.3 
PRIMARY SAFETY ENDPOINTS.............................................................................................................................. 11 
4 
SUBJECT SELECTION AND WITHDRAWAL .................................................................................. 12 
4.1 
INCLUSION CRITERIA .............................................................................................................................................. 12 
4.2 
EXCLUSION CRITERIA ............................................................................................................................................ 12 
4.3 
SUBJECT RECRUITMENT AND SCREENING ......................................................................................................... 12 
4.4 
EARLY WITHDRAWAL OF SUBJECTS .................................................................................................................... 13 
4.4.1 
When and How to Withdraw Subjects ................................................................................... 13 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects ........................................................ 13 
5 
STUDY DRUG .................................................................................................................................... 13 
5.1 
DESCRIPTION .......................................................................................................................................................... 13 
5.2 
TREATMENT REGIMEN ........................................................................................................................................... 13 
5.3 
METHOD FOR ASSIGNING SUBJECTS TOTREATMENT GROUPS ..................................................................... 14 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ................................................................................... 14 
5.5 
SUBJECT COMPLIANCE MONITORING ................................................................................................................. 14 
5.6 
PRIOR AND CONCOMITANT THERAPY.................................................................................................................. 14 
5.7 
PACKAGING ............................................................................................................................................................. 14 
5.8 
BLINDING OF STUDY DRUG ................................................................................................................................... 15 
5.9 
RECEIVING, STORAGE, DISPENSINGAND RETURN ........................................................................................... 15 
5.9.1 
Receipt of Drug Supplies ....................................................................................................... 15 
5.9.2 
Dispensing of Study Drug ...................................................................................................... 15 
5.9.3 
Return or Destruction of Study Drug ..................................................................................... 15 
6 
STUDY PROCEDURES ..................................................................................................................... 16 
6.1 
VISITS 1, 8, & 11 (BASELINE AND FOLLOW-UP ASSESSMENT VISITS) ................................................ 16 
6.2 
VISITS 2, 4, 6, 7, 9, & 10 (COMPLIANCE/SAFETY PHONE CONTACTS) ................................................ 16 
6.3 
RETURN ASSESSMENT VISIT ................................................................................................................ 17 
6.4 
SECOND RETURN VISIT ......................................................................................................................... 17 
7 
STATISTICAL PLAN ......................................................................................................................... 18 
7.1 
SAMPLE SIZE DETERMINATION............................................................................................................................. 18 
7.2 
STATISTICAL METHODS ......................................................................................................................................... 19 
7.3 
SUBJECT POPULATION(S) FOR ANALYSIS .......................................................................................................... 19 
8 
SAFETY AND ADVERSE EVENTS .................................................................................................. 19 
8.1 
DEFINITIONS ............................................................................................................................................................ 19 
8.2 
RECORDING OF ADVERSE EVENTS ..................................................................................................................... 21 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS ..................................................................................................... 21 
8.3.1 
Study Sponsor Notification by Investigator ........................................................................... 21 
8.3.2 
IRB Notification by Investigator ............................................................................................. 22 
8.3.3 
FDA Notification by Sponsor ................................................................................................. 22 
8.4 
UNBLINDING PROCEDURES................................................................................................................................... 22 


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8.5 
STOPPING RULES ................................................................................................................................................... 22 


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8.6 
MEDICAL MONITORING .......................................................................................................................................... 22 
8.6.1 
Internal Data and Safety Monitoring Board ........................................................................... 23 
8.6.2 
Independent Data and Safety Monitoring Board ................................................................... 23 
9 
DATA HANDLING AND RECORD KEEPING ................................................................................... 23 
9.1 
CONFIDENTIALITY ................................................................................................................................................... 23 
9.2 
SOURCE DOCUMENTS ........................................................................................................................................... 24 
9.3 
CASE REPORT FORMS ........................................................................................................................................... 24 
9.4 
RECORDS RETENTION ........................................................................................................................................... 24 
10 
STUDY MONITORING, AUDITING, AND INSPECTING .................................................................. 25 
10.1 
STUDY MONITORING PLAN .................................................................................................................................... 25 
10.2 
AUDITING AND INSPECTING ................................................................................................................................... 25 
11 
ETHICAL CONSIDERATIONS .......................................................................................................... 25 
12 
STUDY FINANCES ............................................................................................................................ 26 
12.1 
FUNDING SOURCE .................................................................................................................................................. 26 
12.2 
CONFLICT OF INTEREST ......................................................................................................................................... 26 
12.3 
SUBJECT STIPENDS OR PAYMENTS ..................................................................................................................... 26 
13 
PUBLICATION PLAN ........................................................................................................................ 26 
14 
REFERENCES ................................................................................................................................... 26 
15 
ATTACHMENTS ................................................................................................................................ 33 


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List of Abbreviations 
 
 
CFR 
Code of Federal Regulations 
CNBD 
Center for Neurobehavioral Development 
CRF 
Case Report Form 
FAS 
Fetal Alcohol Syndrome 
FASD 
Fetal Alcohol Spectrum Disorders 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GRAS 
Generally Recognized as Safe 
ICH 
International Conference on Harmonization 
IRB 
Institutional Review Board 
RDI 
Recommended Dietary Intake 


Clinical Research Protocol Template 
Page 1 
 
Version June 30, 2015 
 
 
Study Summary 
 
Title 
Choline Supplementation as a Neurodevelopmental Intervention 
in Fetal Alcohol Spectrum Disorders 
Short Title 
Choline supplementation in FASD. 
Protocol Number 
1R01AA024123 
Phase 
Phase I and II 
Methodology 
Randomized, double-blind, placebo-controlled 
Study Duration 
Five years 
Study Center(s) 
Single-center 
Objectives 
To evaluate the feasibility and tolerability of post-natal choline 
supplementation in young children 
Number of 
Subjects 
60 
Diagnosis and 
Main Inclusion 
Criteria 
Fetal alcohol spectrum disorder; documented prenatal alcohol 
exposure 
Study Product, 
Dose, Route, 
Regimen 
Choline bitartrate,19 mg per kg body weight (typical range: 200- 
425 mg; max dose 500 mg), oral solution administered once daily 
Duration of 
administration 
Nine months 
Reference therapy 
Placebo 
Statistical 
Methodology 
Mixed models growth curve analyses of the outcome measures 
using and Intention to Treat (ITT) approach 


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1. Introduction 
This document is a protocol for a human research study. This study will be conducted 
according to US and international standards of Good Clinical Practice (FDA Title 21 part 
312 and International Conference on Harmonization guidelines), applicable government 
regulations and Institutional research policies and procedures. 
 
1.1 Background 
Fetal Alcohol Syndrome (FAS) is the largest single known cause of mental retardation 
and developmental disability (Abel, 1995). Estimates suggest that the incidence of FAS 
is nearly 1 per 1000 live births in the general population and 4.3% among heavy 
drinkers (Abel, 1995; May & Gossage, 2001). Perhaps more importantly, the incidence 
of the wider spectrum of disorders associated with prenatal alcohol exposure (FASD) is 
thought to be as high as .9% of the general population (Sampson et al., 1997). At 
present, a diagnosis of FAS is typically made with consideration of four criteria: 1. 
prenatal alcohol exposure (documented or not documented); 2. dysmorphic facial 
features; 3. growth deficiency; 4. central nervous system dysfunction (Hoyme et al., 
2005; K. L. Jones & Smith, 1973). However, it is well-established that most prenatally 
exposed individuals do not display the physical characteristics of FAS, but many do 
have structural brain anomalies and neurocognitive impairment (Bookstein et al., 2001; 
Burden et al., 2005b; Mattson et al., 1998; Sowell et al., 
2001a). 
 
As it has become clear that alcohol exposure has serious and permanent adverse 
effects on cognitive, behavioral, emotional, and social development, even without 
producing the complete FAS syndrome, the disorder has been reframed numerous 
times in recognition of various “partial” FAS conditions including Fetal Alcohol Effects or 
FAE (Clarren & Smith, 1978) and Alcohol-Related Neurobehavioral Disorder or ARND 
(Institute of Medicine, 1996). Currently, the term Fetal Alcohol Spectrum Disorders 
(FASD) is now commonly used to describe the entire spectrum of disability resulting 
from prenatal alcohol exposure (Riley & McGee, 2005; Warren et al., 2004). This FASD 
continuum includes, by some estimates, 5 to 15 times as many children as those 
diagnosed with full-criteria FAS (Lupton et al., 2004; May & Gossage, 2001; Sokol et al., 
1980). When one considers the estimates of the US economic impact, which are in the 
billions of dollars per year (Lupton et al., 2004; Rice et al., 1991), and the fact that FAS 
represents only the tip of the iceberg, the staggering nature of the public health problem 
posed by the full range of FASD becomes apparent. 
 
At present, the literature on interventions for individuals with Fetal Alcohol Spectrum 
Disorders (FASD) is very limited, with only two published randomized controlled trials 
(Premji et al., 2007). However, there are promising new treatment options that have not 
been applied in humans. Specifically, recent pre-clinical studies have shown that dietary 
choline supplementation prenatally and even postnatally, as late as days 21-30 in the 
rodent (equivalent to human childhood), attenuates the memory and behavioral deficits 
associated with prenatal alcohol exposure (J. D. Thomas et al., 2007; J. D. Thomas et 
al., 2000). Perinatal choline levels directly influence brain development (Zeisel & 


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Niculescu, 2006) and, thus, a human clinical trial involving choline supplementation is 
warranted. The fact that postnatal choline supplementation is effective in rats as late as 
21-30 days is particularly relevant because FASD is typically only diagnosed later in 
childhood. As no human choline supplementation studies have been done in children 
with prenatal alcohol exposure, it is not known if supplementation will be effective 
postnatally, prenatally, or both. Based on the time periods in which 
choline is effective in pre-clinical models of FASD, and on the fact that the first years of 
human life represent a period of intense brain development, choline supplementation in 
young children appears to have significant potential as an intervention for 
neurodevelopmental disorders including FASD. 
 
1.2 Investigational Agent 
Although the human body produces choline, the demand cannot be met entirely 
endogenously and thus, some choline must be consumed in food. Human cells require 
choline and, without it, die by apoptosis (Albright et al., 1998; Eagle, 1955; Zeisel et al., 
1997). Choline has been classified as an essential nutrient, and guidelines for 
“adequate intake” of choline have been established for infants, children, and adults 
(Food and Nutrition Board - Institute of Medicine, 1998). The guidelines also identify 
adequate intake levels specifically for pregnant and lactating women because of the 
recognized importance of choline to fetal / neonatal development. A recent study 
suggests that daily consumption by adults in the United States (mean = 312 mg. per day 
for men and 314 mg. per day for women) is significantly below the recommended 
‘adequate daily intake’ of 550 mg. per day for men and 425 mg. per day for women 
(Cho et al., 2006). Choline is found in many foods but it has only been within the last 
few years that the specific choline content of various foods and food products was 
published (Zeisel et al., 2003). Foods highest in choline include eggs, liver, nuts, fish, 
and soy. Choline is also considered part of the Vitamin B-complex. 
 
Choline exists in a free form or esterfied in one of several phospholipid forms including 
phosphatidylcholine, glycerol-phosphocholine and sphingomyelin. It is also found in the 
neurotransmitter acetylcholine. As a precursor to acetylcholine, phospholipids, and 
betaine, choline is critical to the formation of cell membranes, transmembrane signaling, 
cholinergic neurotransmission, and the metabolism of lipids and cholesterol (Food and 
Nutrition Board - Institute of Medicine, 1998). In the context of brain development, 
choline is intricately linked to another essential nutrient, folate, and deficiencies in either 
nutrient lead to disruptions in neural tube closure (Shaw et al., 2004; Smithells et al., 
1976; Zeisel, 2009b). Historical experiences with folate, iron, and iodine fortification 
have dramatically demonstrated the importance of essential nutrients during periods of 
development. For example, when cereal grains were first fortified with folic acid, the 
rates of neural tube birth defects dropped by 26% (Centers for Disease Control and 
Prevention, 2004). Although choline deficiency during pregnancy likely has less 
dramatic effects than folate deficiency, very little is actually known about the impact of 
deficiency or supplementation during critical developmental periods. 


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1.3 Preclinical Data 
Pre-clinical studies have demonstrated that: 
 
1. Perinatal choline deficiency results in abnormal brain development and cognitive 
impairment (Albright et al., 1999b; Holmes-McNary et al., 1997; Wong-Goodrich et al., 
2008) and 2. Perinatal choline supplementation contributes to structural brain changes 
(Li et al., 2004) and enhanced performance on measures of cognition including memory 
(Cheng et al., 2008; Meck et al.,1988; Meck & Williams, 1997a). Choline deprivation in 
rats leads to a 4-fold increase in the rate of neural tube defects (Shaw et al., 2004). In 
addition, choline deficiency leads to permanent structural and biochemical brain 
abnormalities that may be related to underlying changes in neurogenesis and 
differentiation in fetal hippocampus and septum, areas of brain that are critical for 
normal spatial learning and memory (Cermak et al., 1999). Furthermore, prenatal 
choline supplementation leads to enhanced visual-spatial memory performance (Meck 
et al., 1988, 1989; Meck & Williams, 1997a, 1997b) and reductions in proactive 
interference during learning (Meck & Williams, 1999). 
 
Perinatal choline levels have an impact at multiple levels of neurodevelopment. Choline 
contributes to increased dendritic arborization in hippocampal CA1 cells, larger somata, 
and critical functional changes to these cells (J. P. Jones et al., 1999; Li et al., 2004; 
Mellott et al., 2004; Montoya et al., 2000; Pyapali et al.,1998). Prenatal and postnatal 
choline supplementation also affects choline acetyltransferase levels in both 
hippocampus and frontal cortex in rats which, in turn, is associated with improved 
memory functioning, especially in visual-spatial memory (Meck et al., 1989; Williams et 
al., 1998). It has also been demonstrated that the ability of cholinergic neurons to 
produce acetylcholine is directly related to the availability of free choline, its precursor 
(Blusztajn & Wurtman, 1983). Furthermore, choline levels have an impact throughout 
the brain on apoptosis, the programmed cell death that is part of normal brain 
development (Holmes-McNary et al., 1997; Yen et al., 2001). In addition, choline is 
required for the production of a number of phopholipids (such as phosphatidylcholine, 
sphingomyelin, and plasmalogen) that are present in all cells including neurons. These 
membrane phospholipids are necessary for axonal growth and myelination, among 
other processes (Zeisel & Niculescu, 2006). Thus, the evidence suggests that the 
availability of free choline has important effects, depending on discrete time windows, 
on basic aspects of brain structure and function in the developing brain (J. P. Jones et 
al., 1999). 
 
Importantly, pre-clinical choline studies provide strong evidence that choline levels 
during critical periods of brain development have a permanent impact on brain structure 
and function throughout the lifespan. For example, electrophysiological studies of 
hippocampal CA1 cells in brain slices of adult rats that were either deprived of choline 
or supplemented with choline during gestation show permanent changes in long term 
potentiation (LTP) (J. P. Jones et al., 1999). LTP reflects an increase in strength of the 
synaptic connection between neurons following high frequency stimulation (Bliss & 
Gardner-Medwin, 1973; Bliss & Lomo, 1973) and is thought to be an important 
substrate of learning and memory (Bliss et al., 2003). Meck and colleagues have 


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described the impact of perinatal choline availability on brain structure as “metabolic 
imprinting” because of the permanent changes that occur in the cholinergic system, 
affecting brain functioning throughout the lifetime (Meck & Williams, 2003). 
 
Perinatal choline supplementation attenuates the negative effects of prenatal alcohol 
exposure. Thomas et al. (2000) initially demonstrated that administering choline to 
prenatally-exposed rat pups on postnatal days 2-21 resulted in improved performance 
on a discrimination task and a delayed discrimination task. Without postnatal choline 
supplementation, the alcohol-exposed rats were impaired on both tasks. Interestingly, 
choline improved the performance of a non-alcohol-exposed group as well, but the 
largest effects were seen in the alcohol-exposed rats, perhaps because they had the 
most room for improvement. In this study, as in others, the beneficial effects of choline 
on cognitive functioning continued (at least until postnatal day 45 when testing 
occurred) long after choline administration had been discontinued (post-natal day 21). A 
number of other studies have shown similar effects of postnatal choline improving 
cognitive functioning including visual-spatial learning, spatial reversal learning, and fear 
conditioning among rats prenatally exposed to alcohol (Wagner & Hunt, 2006). In 
addition, choline supplementation during postnatal days 4-30 has also been shown to 
reduce excess activity of rats in an open-field situation (J. D. Thomas et al., 2004). This 
finding is intriguing because prenatal alcohol exposure causes postnatal hyperactivity in 
rats, and is associated with hyperactivity and inattention in humans with FASD. 
 
In normally developing rats, the “window” for the beneficial effects of choline appears to 
include gestation and the first 30 postnatal days with the most significant benefits 
obtained if choline supplementation is given during both prenatal and postnatal periods 
(Meck et al., 1989). However, the critical windows for choline’s effectiveness may in fact 
be different for animals whose brains have been damaged by prenatal alcohol 
exposure. Ryan et al. (2008) demonstrated that both “early” postnatal choline 
supplementation (postnatal days 11-20) and “late” choline supplementation postnatal 
days 21-30) significantly attenuated the cognitive deficits induced by prenatal alcohol 
exposure. There was a slight advantage for the early supplementation, but it was not a 
large difference. Thus, the authors concluded that “there is not a strict temporal window 
for choline to be effective, at least on some behavioral measures” and that “the 
developmental window for choline to be effective is either quite large, or spans between 
the PD (postnatal days) 11-20 and 21-30 time periods.” 
Overall, these results suggest that, in humans, choline supplementation could attenuate 
the negative effects of prenatal alcohol exposure across a relatively wide period of 
postnatal development, perhaps even across the first 5 to 10 years of life. 
 
Extrapolating from the pre-clinical trial findings of choline’s efficacy to potential 
applications in humans is challenging due in part to the problems of cross-species 
neurodevelopmental comparisons. Thomas et al.’s studies in FASD (J. D. Thomas et 
al., 2007; J. D. Thomas et al., 2004; J. D. Thomas et al., 2000) and much of the pre- 
clinical work in “normally developing” animals has been done with rats (Meck et al., 
1988, 1989; Meck & Williams, 1997a). Fortunately, there have been significant 
advances in our understanding of cross-species comparisons (Clancy et al., 2001; 


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Clancy et al., 2007) and some have argued that the prenatal and early postnatal periods 
are the most reliable time period during the lifespan in which to make such comparisons 
because of the remarkable similarity in timing and order of critical neurodevelopmental 
events across species (Finlay & Darlington, 1995). This may be especially true with 
regard to specific neural systems or structures. 
 
As an example, we know a great deal about the timing and sequencing of hippocampal 
development in rats and humans (Avishai-Eliner et al., 2002). In humans, there is a 
rapid increase in hippocampal development during the first two years of life, followed by 
a period of slow, steady growth until adolescence (Utsunomiya et al.,1999). Critical 
aspects of differentiation and synaptogenesis are occurring in the hippocampus 
between 3 and 5 years of age. This rapid development includes maturation of pyramidal 
cell dendrites in CA3. The equivalent developmental maturation in rats takes place by 
21 postnatal days, within the timeframe of choline’s efficacy in pre-clinical studies. 
Similarly, in humans, hippocampal-dependent learning and memory processes continue 
to mature until 4 or 5 years of age. The equivalent developmental maturation of memory 
processes in the rat occurs on postnatal days 15 and 16, again within the timeframe of 
choline’s efficacy. MRI volumetric studies have now demonstrated that the human 
hippocampus undergoes a rapid growth spurt in the first two years of life and continues 
a slow, steady growth throughout childhood. 
 
The principal of clinical equipoise, describing the state of genuine uncertainty in the 
expert medical community about the potential efficacy of an intervention (Freedman, 
1987), is an important factor in decisions about the ethics and practicality of a 
randomized clinical trial. At the present time, despite impressive pre-clinical evidence, 
there is very little human data and no consensus about the potential 
neurodevelopmental benefits of postnatal choline supplementation in humans 
(Cockburn, 2003). This uncertainty supports the need for a comprehensive trial. 
 
The ability of choline supplementation to modify certain aspects of brain development 
and mediate the effects of damage during development may prove useful in other 
neurodevelopmental disorders. For example, Holmes et al. (2002) have shown that 
prenatal choline supplementation of rats protects them from cognitive impairment 
typically seen in induced-seizures at a point much later in development. This work, and 
other similar work on choline supplementation suggests that nutritional factors during 
critical periods of brain development (prenatal and postnatal) has long-lasting effects on 
neuroplasticity (Williams, 2008). Intriguingly, the effects of early supplementation appear 
to last throughout the lifetime of the organism. For example Glenn et al. (2008) found 
that prenatal choline supplementation in rats on embryonic days 12-17 led to less age- 
related decline in cognitive functioning (open field exploratory behavior) at 25 months 
(considered old age in rats). This life-long protective effect of choline was associated 
with increased numbers of newly proliferated hippocampal cells as well as increased 
levels of vascular endothelial growth factor and neurotrophin-3 in the brains of the rats 
who received prenatal choline. 


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In addition, Nag et al. have recently demonstrated several neurodevelopmental benefits 
of postnatal choline supplementation in a rat model of Rett Syndrome (Mecp2(1lox) 
mice) including improved locomotion (Nag & Berger-Sweeney, 2007) and increased 
striatal nerve growth factor (Nag et al., 2008). 
 
1.4 Clinical Data to Date 
Dr. Steven Zeisel, Professor of Nutrition and Pediatrics at the University of North 
Carolina and director of the Nutrition Research Institute (nri.unc.edu) and a consultant 
on this project, is one of the world’s leading nutrition experts. Dr. Zeisel has been 
studying choline for more than 30 years and has been responsible for many of the most 
important findings including the studies initially demonstrating that choline is an 
essential nutrient in humans (Sheard & Zeisel, 1986; Zeisel et al., 1991). Dr. Zeisel’s 
work has identified choline levels in common foods (Howe et al., 2004), identified 
genetic polymorphisms associated with choline metabolism (daCosta et al., 2006; 
Zeisel, 2007, 2008), and investigated relationships between choline and numerous 
human diseases (Albright et al., 1997; Bidulescu et al., 2007; Cho et al., 2007; Xu et al., 
2008; Zeisel, 1992). Most relevant to the current proposal, Dr. Zeisel has demonstrated 
the role of choline in normal brain development as well as the potential for choline 
supplementation during critical periods of brain development (Conlay & Zeisel, 1982; 
Magil et al., 1981; Sanders & Zeisel, 2007; Zeisel, 1997, 2000, 2004, 2006, 2009a; 
Zeisel & Niculescu, 2006). 
 
Our research group has recently completed the first pilot studies to evaluate choline 
supplementation as a neurodevelopmental intervention in children with FASD. We 
first conducted a double- blind, randomized, placebo-controlled pilot study 
(R21AA019580) that focused on establishing the safety and tolerability of choline 
in children with FASD (n=20) (Wozniak, Fuglestad et al. 2013). That study evaluated 
the safety of choline (there were no serious adverse events) and demonstrated high 
tolerability (children received partial or full doses on 87% of 270 days in the study). 
We recently completed a larger double-blind, randomized, placebo controlled 
trial that included a total of 40 additional participants, ages 2.5 to 5 years 
(R33AA019580). Compared to placebo, choline supplementation improved 2-3 year old 
children’s performance on an elicited imitation paradigm, a hippocampus-dependent 
memory task. These data are outlined in the preliminary studies section below. The 
results comprise the first available human evidence that the pre-clinical findings in 
rodents may translate to humans. The results from our initial two studies of choline in 
children with FASD directly inform the next phase of this work which will include 1. 
Testing a modified dosing scheme based on the efficacy and side effect data collected 
in the previous trials; 2. Determining the longevity of the cognitive changes attributable 
to choline; 3. Examining the specificity/generalizability of choline’s effects by measuring 
additional cognitive domains; 4. Further evaluating a choline-specific gene as apotential 


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moderator of treatment response. The genomic hypothesis centers around a single 
nucleotide polymorphism (SMP) that is involved in endogenous choline synthesis 
(phosphatidylethanolamine N-methyltransferase (PEMT) rs12325817 SNP). The SNP 
is associated with significantly greater susceptibility to dietary choline insufficiency 
(Fischer, da Costa et al. 2010). The hypothesis is that children with one or two alleles 
of rs12325817 (CG or CC genotype) will show a significantly larger response to choline 
treatment on measures of cognitive functioning compared to those with the normal 
genotype (GG) because they have a lower capacity to synthesize choline 
endogenously. 
 
A series of papers from a Cleveland Clinic research group have raised a potential link 
between trimethylamine-N-oxide or TMAO, a metabolite of choline and carnitine, and 
atherosclerosis in older adults with cardiovascular disease (Wang et al., 2011; Tang et 
al., 2013; Koeth et al., 2013). An initial metabolomic study of patients undergoing 
cardiac evaluation showed an association between plasma TMAO levels and acute 
cardiovascular events. A later study showed a similar relationship between plasma 
TMAO levels in patients undergoing coronary angiography and cardiovascular events 
over a 3-year follow-up period. Another study reported that atherosclerosis-prone 
(APOE--) mice that were fed choline or TMAO showed greater aortic root 
atherosclerotic plaque compared to control animals. In contrast, a study in hamsters 
demonstrated an inverse relationship between plasma TMAO and atherosclerosis 
(Martin et al., 2009). Two large studies of cardiovascular risk factors in humans, the 
Atherosclerosis Risk in Communities (14,430 men and women) study (Bidulescu et al., 
2007) and the PROSPECT-European Prospective Investigation into Cancer and 
Nutrition (16,165 women) study (Dalmeijer et al., 2008) observed no significant increase 
in cardiovascular risk with increasing dietary intake of choline. An editorial 
accompanying the Tang et al. (2013) paper in the New England Journal of Medicine 
highlights the importance of further examining the new model but also cautions “These 
speculative comments and suggestive observations indicate that much remains to be 
done to determine the precise role of TMAO in atherothrombogenesis — whether it has 
a direct effect on pathogenesis, is an epiphenomenal biomarker, or is a precursor to a 
more direct effector.” (Loscalzo at al., 2013). In summary, these emerging data are 
important but do not imply a clear risk to healthy youhg individuals taking choline 
supplementation. We have informed the FDA of these developments in the literature 
with an Administrative Supplement and we will continue to monitor TMAO levels in the 
trial in order to provide additional data to the field. 
 
1.5 Dose Rationale and Risk/Benefits 
For children ages 1 to 3 years, the recommended dietary intake (RDI) for choline is 200 
mg. and the maximum upper limit for tolerability is 1000 mg. (Food and Nutrition Board - 
Institute of Medicine, 1998). For children ages 4 to 8 years, the RDI for choline is 250 
mg. and the maximum upper limit for tolerability is also 1000 mg. (eating two eggs 
would provide an equivalent of 545 mg. of choline). The goal of the current study is to 
provide choline supplementation as opposed to simply ensuring choline sufficiency. 
Our previous two studies used a fixed 500 mg. dose, regardless of the child’s weight 


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(Wozniak et al. 2013, and Wozniak et al., 2015). The dose was well-tolerated, with 
partial or full doses received on 88% of the 270 days in the study. There was no 
difference between 500 mg. choline and placebo in terms of doses received or 
participant drop-out. There were no serious adverse events and no adverse events that 
differed by group other than fishy body odor in the choline group (an expected adverse 
event). A substantial proportion of children in the choline group (52%) compared to the 
control group (4%) did experience fishy body odor at some point in the 9 month study. 
For the smallest children in the study by weight, nearly 100% experienced fishy body 
odor at some point. 
 
For the current study, choline will be given at a 19 mg per kg dose, based on evidence 
from the prior two pilot studies showing increased rates of fishy body odor at higher 
doses and also on data indicating that memory performance was optimal at doses in the 
19 mg per kg range.  Choline will be administered in the form of a fruit-flavored drink 
mix containing choline bitartrate (compounded for the study by Fagron, St. Paul, MN; 
Investigational New Drug Application #107085). Balchem (New Hampton, NY) 
manufactures the choline bitartrate to USP standards.  The parent will add the powder 
to plain water. The matching placebo drink mix (without choline bitartrate) will also be 
provided by Fagron. Material will be stored, labeled, allocated, and dispensed by the 
University of Minnesota’s Investigational Drug Service (IDS) Pharmacy. 
 
Participant families will be given a 65 day supply initially. Re-supply will be handled by 
packages mailed after the investigators have verified the participant’s continued 
involvement in the study and freedom from serious adverse events during telephone 
visits. The preparation, coding, and tracking will be handled by the University of 
Minnesota’s Investigational Drug Service pharmacy. 
 
Choline is considered a GRAS substance (generally regarded as safe) by the FDA and 
the dose to be utilized in this study is lower than the maximum tolerable upper limit. 
Based on our previous study, tolerability for this lower dose of choline is expected to be 
high. Parents will be educated at the time of enrollment about potential side effects of 
choline which rarely occur and usually at much higher doses than those proposed 
(hypotension has been reported at 7.5 grams per day; gastrointestinal symptoms (loss 
of appetite, upset stomach), sweating, or body odor have occurred at 10-16 grams per 
day). As indicated, in the first 60 participants, we only observed fishy body odor and not 
other adverse events. 
 
At one month, a full range of potential side effects will be screened. Although allergic 
reactions to choline are not expected (we observed none in the pilot studies), we will 
monitor for a range of allergic symptoms nonetheless. Adverse events and serious 
adverse events will be reported to the University of Minnesota IRB and to Quorum IRB. 
Serious adverse events will also be reported to the NIH program officer. 


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2. Study Objectives 
 
Primary Objective 
Evaluate the efficacy of choline in treating memory deficits and executive functioning 
deficits in children with FASD. 
 
Secondary Objectives 
Evaluate the long-term effects of choline supplementation by re-assessing children who 
took choline and placebo as part of the previous pilot study. 
 
Evaluate the potential interaction between genotype ((for example PEMT, rs12325817) 
and response to choline intervention. 
 
3. Study Design 
3.1 General Design 
This is a randomized, double-blind, placebo-controlled study. Sixty children, male and 
female, between the ages of 2 years and 5 years at enrollment will be included. 
 
Eligible participants will be randomized to treatment or placebo in a 1:1 allocation ratio. 
A block randomization procedure with variable block sizes will be used to maximize 
unpredictability of assignment. 
 
Participants will receive the test article for nine months and will have an in-person 
baseline visit followed by 11 additional visits (9 telephone visits; 2 additional in-person 
visits) for a total of 12 visits. A telephone visit for purposes of safety and compliance will 
occur at 2 weeks. A telephone visit will be scheduled for 1 month after administration of 
the test article begins. Formal complete follow-up assessments will occur at 6 months 
and 9 months. Telephone visits for safety and compliance will occur at months 2, 3, 4, 
5, 7, 8 and 10. Parent-report measures of behavior will be collected. Behavioral 
paradigms will be used to assess memory and other cognitive functions. Formal 
psychometric testing to establish developmental levels will be completed. Blood will be 
collected to assess compliance with the treatment and effect on blood choline levels. 
DNA will also be extracted for analysis of single nucleotide polymorphisms related to 
endogenous choline production. Plasma will also be analyzed for immune function 
and micro-RNA. 
 
As part of the project, participants from the previous study (1R21AA019580 / 
4R33AA019580) will be brought back to the University for a set of cognitive evaluations 
to assess the long-term effects of having taken choline/placebo previously. 
 
3.2 Primary Study Endpoints 
The primary study endpoints will be cognitive assessments. These will include the 
Elicited Imitation memory task and the Stanford-Binet Intelligence Scales. 


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3.3 Secondary Study Endpoints 
Secondary cognitive endpoints will include the Minnesota Executive Functioning Scale 
(Carlson, 2014), and the NIH Toolbox Flanker Inhibitory Control and Attention Task 
(MacDonald, 2014). We will also evaluate potential behavioral and/or emotional 
changes with a caregiver-report paper and pencil measure (Child Behavior Checklist). 
 
We will also evaluate feasibility and tolerability by collecting empty and full choline 
packages to monitor compliance. Serum choline levels will be obtained. 
 
As part of the project, we will bring up to 40 participants (20 choline and 20 placebo) 
from our previous study (1R21AA019580 / 4R33AA019580) back to the University for a 
brief visit focused on examining the potential long-term cognitive effects of having taken 
choline or placebo. All who participated in the previous study will be eligible for the 
return visit. No study drug will be used at this visit and no blood will be collected. The 
measures administered will be cognitive outcome measures. The Elicited Imitation 
paradigm, used in the initial study, will be repeated using materials/items that have not 
previously been seen by the child. The Stanford Binet Intelligence Scales will be 
administered. The NIH Toolbox Picture Sequence Memory Test is a measure of 
episodic memory appropriate for ages 3 and up (McDonald 2014). The NIH 
Dimensional Card Sort Task is a measure of executive functioning from the same NIH 
Toolbox. The NIH Flanker Task is a measure of attentional control from the same NIH 
toolbox. The Narrative Memory subtest from the NEPSY-II is a measure of verbal 
memory for ages 3 to 16 (Korkman, Kirk et al. 2007). The Memory for Names subtest is 
another verbal memory subtest from the NEPSY-II. The child’s height and weight will 
also be measured. 
 
We will also bring up to 20 participants (10 choline and 10 placebo) from our previous 
study (1R21AA019580 / 4R33AA019580) back to the University for a neuroimaging 
visit focused on examining the potential long-term brain development effects of having 
taken choline or placebo. Participants will undergo MRI scans and brief neurocognitive 
testing at this follow-up visit. 
 
3.4 Primary Safety Endpoints 
No safety endpoints have been defined. Choline is a normal component of the human 
diet, so there are relatively few risks associated with supplementation. The doses to be 
used in the proposed project are below the upper tolerable limit for choline (1000 mg 
per day) and at or below the doses previously used in our pilot studies. At much higher 
doses than we are proposing, choline supplementation has been associated with mild 
hypotension. We observed no evidence of hypotension in 60 participants in the 
previous pilot studies. In the literature, some individuals report gastrointestinal 
disturbance associated with very high levels of choline supplementation. We observed 
no significant increase in GI symptoms with choline in 60 participants during the 
previous pilot studies. A full range of potential side effects will be screened. 
 
Although allergic reactions to choline are not expected, we will monitor for a range of 
allergic symptoms nonetheless. There are no reported manifestations of intoxication of 
any kind nor overdose from the choline preparation to be used in this study. No toxic 


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effects have been reported. 
 
NOTE that a Data and Safety Monitoring Board (DSMB) is in place and will review the 
study progress and adverse events. 
 
4. Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
1. Ages 2.5 years to 5 years of age* 
2. Available parent or legal guardian capable of giving informed consent for 
participation. Participation of children under guardianship (wards of the state) 
needs to be authorized by a court order. 
3. Modified Institute of Medicine (IOM) criteria for Fetal Alcohol Syndrome (FAS), 
Partial Fetal Alcohol Syndrome (PFAS), or Alcohol-Related Neurodevelopmental 
Disorder (ARND) (Hoyme, May, et al., 2005). 
 
4.2 Exclusion Criteria 
1. Known history of a neurological condition (ex. epilepsy, traumatic brain injury) 
2. Known history of a medical condition known to affect brain function. 
3. Known history of other neurodevelopmental disorder (ex. autism, Down 
syndrome) 
4. Known history of very low birthweight (<1500 grams) 
 
*For the follow-up studies of returning participants from the previous study 
(1R21AA019580 / 4R33AA019580), the inclusion/exclusion criteria will be the same 
as above except that the age range will be extended to incorporate the fact that the 
children are now older. The inclusion/exclusion criteria for the follow-up study will be 
from 2.5 to 15 years of age. 
 
4.3 Subject Recruitment and Screening 
Participants will be recruited from two University of Minnesota Medical Center Clinics in 
which co-investigators see patients: The Fetal Alcohol Spectrum Disorders clinic (Dr. 
Christopher Boys, Ph.D., Dr. Amy Gross, Ph.D) and the International Adoption Clinic 
(directed by Dr. Judith Eckerle, M.D.). Families of potential participants will be 
contacted by research staff. Visitors to these clinics may also see flyers and brochures 
describing the study at the time of their clinic appointment although they will not be 
recruited directly by their clinician. Mailings will also be sent to families of children with 
FASD via lists provided by the Minnesota Organization on Fetal Alcohol Syndrome 
(MOFAS). MOFAS has close working relationships with Dr. Wozniak, Dr. Eckerle, Dr. 
Boys, Dr. Gross and both clinics. All recruitment materials will be submitted for IRB 
approval upon development, before use in the study. Telephone scripts will be utilized. 
 
For the follow-up assessment, all participants from the previous study (1R21AA019580 / 
4R33AA019580) will be invited to participate. Contact will be by letter and/or telephone 
call. Scripts will be utilized. 


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4.4 Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
Participants may withdraw from the study at any time. The investigators may initiate the 
withdrawal of a study participant if significant side effects are apparent such as major 
gastrointestinal disturbance, diarrhea, hypotension, or allergic reaction. 
 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
At the time of enrollment, contact information will be obtained for the participating family 
including address, home telephone number, cell phone number, and email address. 
We may also request contact information for a secondary caregiver of the child who 
would know their status in the future in case the study loses contact with the parent / 
primary caregiver. We will attempt to collect some follow-up information at 9 months for 
all withdrawn subjects (the normal point of study conclusion). If the withdrawn 
participant family is willing to be interviewed in person or by telephone about the child, 
we will collect side effect information, other health information such as the emergence of 
new medical problems, behavioral data (via the standard study behavioral rating 
instruments), and information about the reasons for study withdrawal. 
 
5. Study Drug 
5.1 Description 
Choline, which is considered part of the Vitamin B-complex, is an essential nutrient 
normally consumed on a daily basis in many foods. Supplemental choline will be 
administered in the form of a fruit-flavored drink mix containing choline bitartrate 
(compounded for the study by Fagron, St. Paul, MN under IND#107085). Balchem 
(New Hampton, NY) manufactures the choline bitartrate to USP standards. The parent 
will add the powder to plain water. The matching placebo drink mix (without choline 
bitartrate) will also be provided by Fagron. Material will be stored, labeled, allocated, 
and dispensed by the University of Minnesota’s Investigational Drug Service (IDS) 
Pharmacy. 
 
5.2 Treatment Regimen 
Choline will be administered at a dose of 19 mg per kg once daily, as an oral solution for 
nine months. Placebo will be administered as an oral solution once daily for nine 
months. Choline will be administered under FDA Investigational New Drug Application 
#107085. Parents will be informed that choline is being regulated as an investigational 
drug for purposes of this clinical trial. 


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5.3 Method for Assigning Subjects to Treatment Groups 
Randomization will be managed by using a computer procedure for randomization and 
tracking. A block randomization with variable block sizes will be used to maximize 
unpredictability of assignment. The University of Minnesota Investigational Drug 
Service will have the randomization code and will assign subjects and distribute the test 
article according to the randomization schedule. 
 
5.4 Preparation and Administration of Study Drug 
Fagron will prepare the choline fortified fruit-flavored drink mix and placebo drink mix for 
the study (IND#107085). Participating families will be provided with a supply of the 
material in the form of individual packages. The parent / guardian will prepare the drink 
by adding the powdered drink mix to plain water and will then administer it to the child. 
The parent/guardian will be trained to prepare and administer the test article at the time 
of the baseline visit. 
 
5.5 Subject Compliance Monitoring 
The parent/guardian of each subject will be asked to keep all empty study packets in a 
provided Ziploc bag. Self-addressed, postage-paid large envelopes will be provided to 
participant families to facilitate the return of empty packets. At the time of Visit 8 and 
Visit 11, the parent/guardian will be asked to bring all used and unused packets of the 
test article. Study staff will count the used and unused packets. Information will be 
recorded on the compliance case report form for each follow-up visit. 
 
Significant non-compliance will be reported to the investigator after each follow-up visit. 
Compliance will be discussed and encouraged at each follow-up visit including 
telephone visits. 
 
5.6 Prior and Concomitant Therapy 
At baseline, and at every follow-up visit, the parent/guardian will be asked to provide 
information about any over-the-counter or prescription medications the subject is 
currently taking or has been given since the last visit. The parent/guardian will be asked 
to provide the name of the substance, the dose, the starting date and the stopping date. 
 
After enrollment, the new initiation of nutritional supplements containing choline or 
betaine will be disallowed. All other concomitant therapies, including potential 
behavioral interventions, will be allowed and recorded. 
 
5.7 Packaging 
The test article, a powdered drink mix, will be packaged in individual foil packets. The 
powdered drink mix in each foil pack will contain either choline bitartrate or placebo. 
The test article will be delivered by courier from the manufacturer (Fagron) to the 


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University of Minnesota Investigational Drug Service where the labels will be prepared 
and where the material will be kept prior to distribution to research participants. Each 
foil packet will be labeled with a study number, date of manufacture, a code by which 
the contents (choline bitartrate vs. placebo) can be tracked, cautionary note, and 
indication of “investigational drug” status. 
 
5.8 Blinding of Study Drug 
Each packet of test article will be coded with an arbitrary code number. The University 
of Minnesota Investigational Drug Service will keep the link between these codes and 
the contents (choline bitartrate vs. placebo). The investigators will remain blinded 
throughout the study. The study statistician, Ann Brearley, will have access to the link 
and, therefore, will be unblinded. 
 
5.9 Receiving, Storage, Dispensing and Return 
5.9.1 Receipt of Drug Supplies 
Balchem, the manufacturer of the active agent and the placebo, will ship the product to 
a compounding pharmacy (Fagron). The transportation and packaging will take place 
according to GMP (good manufacturing practices). Fagron will then ship the material in 
sealed foil packets to the University of Minnesota Investigational Drug Service (IDS). 
IDS will perform an inventory, complete a drug receipt log, and return any damaged 
material to the manufacturer. This will be documented and the study sponsor will be 
notified. There are no special storage requirements for this product. 
 
5.9.2 Dispensing of Study Drug 
The Investigational Drug Service will assign the test article (choline bitartrate or 
placebo) to each participant by consulting the randomization schedule. The 
investigators will pick up the coded test article from the IDS in several amounts. Initially, 
30 packets will be distributed to participating family at the study baseline visit. The 
remaining material will be distributed in batches of 60 packets. 
 
The investigator will keep a log onsite to ensure all medication that is dispensed is 
recorded the day of the baseline visit and the days of shipment. 
 
5.9.3 Return or Destruction of Study Drug 
At the completion of the study, there will be a final reconciliation of test article 
distributed, consumed, and remaining. This reconciliation will be logged on the drug 
reconciliation form, signed and dated. Any discrepancies noted will be investigated, 
resolved, and documented prior to destruction of unused study drug. Drug destroyed 
on site will be documented in the study files. 


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6. Study Procedures 
6.1 Visits 1, 8, & 11 (Baseline and follow-up assessment visits) 
Participants and their parent/guardians identified as described in Section 4.3 of this 
protocol will be asked to come for a baseline visit (Visit 1). This visit is expected to last 
for approximately 3 to 3.5 hours. Visits 8 and 11 will be similar, but slightly shorter (2.5 
to 3 hours) because several tasks are not repeated. The following procedures will occur: 
 
• Consenting process (Visit 1 only); 
• Medical history (Visit 1 only) and physical exam 
• Concomitant medications will be recorded 
• Height 
• Weight 
• Head circumference 
• Facial photograph to allow for detailed measurements (Visit 1 only) 
• Blood pressure 
• Blood collection for choline and metabolite (betaine, phosphatidylcholine, 
sphingomyelin, and trimethylamine-N-oxide (TMAO) level (3 ml tube). 
• Plasma extracted from this whole blood will be analyzed for measures of 
immune function (cytokines) and micro-RNA.. 
• Extraction of DNA sample from any blood sample for single nucleotide 
polymorphisms related to endogenous choline synthesis (PEMT rs12325817) 
• Astley & Clarren’s FASD diagnostic evaluation including a photograph of the 
child’s face for computer measurement and analysis (Visit 1 or Visit 3) 
• Stanford Binet Intelligence Scales, Fifth Edition (Visit 1 and 11 only) 
• Elicited Imitation Paradigm, including video-recording to facilitate scoring 
afterward 
• Minnesota EF Scale (MEFS), Early Childhood Version 
• NIH Toolbox Flanker Inhibitory Control and Attention Task 
• Beery Developmental Test of Visual-Motor Integration (VMI) (any visit) 
• Scales of Independent Behavior (SIB) (any visit) 
• Child behavioral questionnaires (CBCL); a parent-report instrument 
• 24-hour Diet interview (for the parent/guardian to complete about the subject) 
• Child Eating Behavior Questionnaire (CEBQ); a parent-report instrument 
• Randomization (Visit 1 only) 
• Training on how to store, mix, and administer the study medication (Visit 1 only) 
• Adverse events will be recorded (Visits 8 & 11 only) 
• Dispense a supply of the test article (30 packages at Visit 1) 
• Provide instructions for returning for the next follow-up visit (Visits 1 & 8 only) 
 
6.2 Visits 2, 3, 4, 5, 6, 7, 9, 10 & 12 (compliance/safety phone contacts) 
At each of these visits, study staff will place a phone call to the participant’s 
parent/guardian. The following information will be collected: 


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• Adverse events will be recorded 
• Concomitant medications will be recorded (Visits 2,3,4,5,6,7,9 & 10) 
• Any challenges with compliance related to administration of the study medication 
or completing the medication diary (Visits 2,3,4,5,6,7,9 & 10) 
 
Visit 2 will occur 14 days after the baseline visit; Visit 3 will occur at Month 1 after 
baseline; Visit 4 will occur at Month 2 after baseline; Visit 5 will occur at Month 3 after 
baseline; Visit 6 will be at Month 4 after baseline; Visit 7 will be at Month 5 after 
baseline; Visit 9 will be at Month 7 after baseline; Visit 10 will be at Month 8; Visit 12 will 
be at Month 10 after baseline (one month after discontinuation of the test article). The 
acceptable visit window for each of these visits is +/- 7 days for Visit 2 and +/-14 days 
for the remainder of the visits. Each phone call is expected to last approximately 15 
minutes. The purpose of Visit 12 is to collect potential adverse event data after the 
participant is no longer on the test agent. 
 
6.3 Return assessment visit (1R21AA019580 / 4R33AA019580) (2B) 
• Consenting – using separate consent form; Assenting if age =>7 years 
• Height 
• Weight 
• Elicited Imitation Paradigm, including video-recording to facilitate scoring 
afterward 
• The NIH Toolbox Picture Sequence Memory Test 
• The NIH Dimensional Card Sort Test 
• The NIH Picture Sequence Memory Test 
• The Narrative Memory subtest from the NEPSY-II 
• Stanford Binet Intelligence Scales, Fifth Edition 
• Child behavioral questionnaires (CBCL) 
• 24-hour Diet interview 
 
6.4 Second Return Visit (1R21AA019580 / 4R33AA019580) (2C) 
• Consenting – using separate consent form; Assenting for those age =>7 years 
• Delis-Kaplan Executive Function System (DKEFS) Trail Making and Color-Word 
Interference subtests 
• NIH Toolbox List Sorting task 
• Wechsler Intelligence Scale for Children – working memory subtests 
• Child Behavior Checklist (CBCL) 
• 1 hour MRI scan 
• Subjects will be scanned at the Center for Magnetic Resonance Research on a 
Siemens Prisma scanner. High resolution structural scans will be collected 
• MRI scans will not be interpreted clinically. Group MRI analysis will be done using 
already published approaches. 
• Use of radiation: NA – MRI does not use ionizing radiation 
• Use of Center for Magnetic Resonance Research: This study uses the University 
of Minnesota’s CMRR and follows all CMRR protocols and procedures. 


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• 
De-identified neuroimaging data will be stored locally on servers supported by the 
Center for Magnetic Resonance (CMRR). 
• 
De-identified cognitive and behavioral data will be stored in a secure, HIPAA- 
compliant RedCap database supported by the University of Minnesota. 
• Data elements to be stored will include processed, de-identified neuroimaging 
results, de-identified neurocognitive test results, and de-identified behavioral 
questionnaire data. 
 
NOTE: If a participant has recently participated in one of our other protocols, and 
there is recent appropriate MRI or neuropsychological data, signed permission will be 
obtained to utilize those data for the purposes of this protocol (in addition to the 
standard consent and assent forms for this study). 
 
NOTE: Missing data for cognitive measures in studies of children with 
neurodevelopmental disorders are common (because of attention deficits, non- 
compliance, etc.). Behavioral checklists are always presented with the option to skip 
items if they are uncomfortable. Therefore, missing cognitive test data and behavioral 
checklist data are within protocol and not considered deviations. 
 
7. Statistical Plan 
7.1 Sample Size Determination 
For purposes of sample size determination, the dependent measure was the percent of 
correct items on delayed recall of the EI task (averaged across two events per 
timepoint). The effect size from our completed trial was d=0.66. A power analysis was 
carried out using Optimal Design Software for Multi-level and Longitudinal Research 
(Version 3.01) to determine the sample size that would be necessary to detect a 
difference in linear change between treatment and placebo groups with this effect size 
(Feingold 2009). The analysis assumed that the data would be analyzed using a mixed 
models repeated measures design for a person-randomized trial, with an effect size 
d=.66, and a two-tailed type I error of 0.05. To achieve 80% power, a sample size of 
58.5 participants (approximately 30 per treatment arm) is necessary. This is consistent 
with the significant group difference that was observed in the pilot trial. The sample size 
for the proposed study will therefore be fixed at 60 children. Combining participant data 


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for some outcome measures from the first set of pilot studies (N=60) and the proposed 
study (N=60) will provide additional power to further evaluate age, sex, and PEMT 
genotype as moderating variables. 
 
7.2 Statistical Methods 
Statistical analyses will utilize an Intention to Treat (ITT) approach. Mixed models 
analyses with growth curve estimates will form the basis of the core analyses. 
 
7.3 Subject Population(s) for Analysis 
Not applicable 
 
8. Safety and Adverse Events 
8.1 Definitions 
8.1.1 Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that develops or 
worsens in severity during the course of the study. Intercurrent illnesses or injuries 
should be regarded as adverse events. Abnormal results of diagnostic procedures are 
considered to be adverse events if the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
8.1.2 Serious Adverse Event 
Adverse events are classified as serious or non-serious. A serious adverse event is 
any AE that is: 
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
 
Important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance. They may jeopardize the subject, and may require 
intervention to prevent one of the other serious outcomes noted above. For example, 
drug overdose or abuse, a seizure that did not result in in-patient hospitalization, or 
intensive treatment of bronchospasm in an emergency department would typically be 
considered serious. 
 
All adverse events that do not meet any of the criteria for serious should be regarded as 
non-serious adverse events. 
8.1.3 Adverse Event Reporting Period 


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The study period during which adverse events must be reported is normally defined as 
the period from the initiation of any study procedures to the end of the study treatment 
follow-up (10 months in the case of this study). 
8.1.4 Preexisting Condition 
A preexisting condition is one that is present at the start of the study. A preexisting 
condition should be recorded as an adverse event if the frequency, intensity, or the 
character of the condition worsens during the study period. 
8.1.5 General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition. At the end of the study, any new clinically significant findings/abnormalities 
that meet the definition of an adverse event must also be recorded and documented as 
an adverse event. 
8.1.6 Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until the events 
are resolved, the subject is lost to follow-up, or the adverse event is otherwise 
explained. At the last scheduled visit, the investigator will instruct each subject to report 
any subsequent event(s) that the subject, or the subject’s personal physician, believes 
might reasonably be related to participation in this study. 
 
8.1.7 Abnormal Laboratory Values 
A clinical laboratory abnormality will be documented as an adverse event if any one of 
the following conditions is met: 
• The laboratory abnormality is not otherwise refuted by a repeat test to confirm 
the abnormality 
• The abnormality suggests a disease and/or organ toxicity 
• The abnormality is of a degree that requires active management; e.g. change of 
dose, discontinuation of the drug, more frequent follow-up assessments, further 
diagnostic investigation, etc. 
 
8.1.8 Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization will be 
documented and reported as a serious adverse event unless specifically instructed 
otherwise in this protocol. Any condition responsible for surgery will be documented as 
an adverse event if the condition meets the criteria for and adverse event. 
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are 
reported as an adverse event in the following circumstances: 
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical 
procedures for a preexisting condition. Surgery should not be reported as an 
outcome of an adverse event if the purpose of the surgery was elective or 
diagnostic and the outcome was uneventful. 


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• Hospitalization or prolonged hospitalization required to allow efficacy 
measurement for the study. 
• Hospitalization or prolonged hospitalization for therapy of the target disease of 
the study, unless it is a worsening or increase in frequency of hospital 
admissions as judged by the clinical investigator. 
 
8.2 Recording of Adverse Events 
At each contact with the participant family, the investigator will seek information about 
adverse events through specific questioning. At each in-person visit, the investigators 
will also directly evaluate for adverse events by examination. Information on all adverse 
events will be recorded immediately in the source document, and also in the appropriate 
adverse event module of the case report form (CRF). Clearly related signs, symptoms, 
and abnormal diagnostic procedures results will be recorded in the source document. 
 
All adverse events occurring during the study period will be recorded. The clinical 
course of each event will be followed until resolution, stabilization, or until it has been 
determined that the study treatment or participation is not the cause. Serious adverse 
events that are still ongoing at the end of the study period will be followed up to 
determine the final outcome. Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study treatment or study 
participation will be recorded and reported immediately. 
 
8.3 Reporting of Serious Adverse Events 
8.3.1 Study Sponsor Notification by Investigator 
A serious adverse event will be reported to the IRB and to the study sponsor (NIH) by 
telephone within 24 hours of the event. A Serious Adverse Event (SAE) form will be 
completed by the investigator and faxed to the IRB and to the study sponsor within 24 
hours. The investigator will keep a copy of this SAE form on file at the study site. 
Report serious adverse events by phone and facsimile to: 
 
Jeffrey R. Wozniak, Ph.D. 
Voice: 612-273-9741 
Pager: 612-899-8941 
Cell: 612-598-0041 
FAX: 612-273-9779 
 
At the time of the initial report, the following information will be provided: 
• Study identifier 
• Study Center 
• Subject number 
• A description of the event 
• Date of onset 
• Current status 
• Whether study treatment was 
discontinued 
• The reason why the event is 
classified as serious 
• Investigator assessment of the 
association between the event 
and study treatment 


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Within the following 48 hours, the investigator will provide further information on the 
serious adverse event in the form of a written narrative. This will include a copy of the 
completed Serious Adverse Event form, and any other diagnostic information that will 
assist the understanding of the event. Significant new information on ongoing serious 
adverse events will be provided promptly to the study sponsor 
 
8.3.2 IRB Notification by Investigator 
Reports of all serious adverse events (including follow-up information) will be submitted 
to the IRB within 10 working days including those that fall under the UPIRTSO 
guidelines. Copies of each report and documentation of IRB notification and receipt will 
be kept in the Clinical Investigator’s binder. 
 
8.3.3 FDA Notification by Sponsor 
The study sponsor will notify the FDA by telephone or by facsimile transmission of any 
unexpected fatal or life-threatening experience associated with the use of the drug as 
soon as possible but no later than 7 calendar days from the sponsor’s original receipt of 
the information. 
 
If a previous adverse event that was not initially deemed reportable is later found to fit 
the criteria for reporting, the study sponsor will submit the adverse event in a written 
report to the FDA as soon as possible, but no later than 15 calendar days from the time 
the determination is made. 
 
8.4 Unblinding Procedures 
The principal investigators, Jeffrey R. Wozniak, Ph.D. and Michael K. Georgieff, M.D. 
will be blinded as will their co-investigators during the course of the study. However, in 
the event of a serious adverse event, the investigators will contact the Investigational 
Drug Service (IDS) within 24 hours to initiate the unblinding of the subject. This will be 
reported in the case report form. A written narrative of the events will be prepared 
within 48 hours. 
 
8.5 Stopping Rules 
The study will be monitored by a local DSMB. Based on the previous study experience, 
the investigational drug involved (choline), and the goals of the study, there will not be 
formal stopping rules based on efficacy or lack thereof. If a statistically significant group 
difference in serious side effects is identified by the DSMB, the study will be stopped. 
 
8.6 Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at 
his/her site. This safety monitoring will include careful assessment and appropriate 
reporting of adverse events as noted above, as well as the construction and 
implementation of a site data and safety-monitoring plan (see section 9 Auditing, 
Monitoring and Inspecting). Medical monitoring will include a regular assessment of the 
number and type of serious adverse events. 


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8.6.1 Internal Data and Safety Monitoring Board 
The Data Safety and Monitoring Board will consist of three local clinical research 
experts / physicians who will review unblinded data. The group will comprise: 
 
Rebecca Shlafer, Ph.D. 
Assistant Professor of General Pediatrics and Adolescent Medicine 
 
Brandon Nathan, M.D. 
Associate Professor of Pediatrics 
 
Claudia Fox, M.D.,MPH 
Assistant Professor of Pediatrics 
 
The DSMB will meet after 25%, 50% and 75% of the data have been collected and will 
be tasked with overseeing patient safety, study conduct, and study progress.  All 
serious adverse events (SAEs) and adverse events (AEs) with a rating of 1 or higher on 
the NIH Common Terminology Criteria for Adverse Events (CTCAE) scale will be 
considered. Ann Brearley, the study statistician who will be unblinded, will prepare 
reports for the DSMB. The reports will contain information about patient accrual rate 
relative to targets, drop-outs, losses to follow-up, SAE counts, and AE counts by 
treatment condition. 
 
The DSMB will record its summary in a formal report to be filed with the University of 
Minnesota IRB and Quorum IRB. 
 
8.6.2 Independent Data and Safety Monitoring Board 
None 
 
9. Data Handling and Record Keeping 
9.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 
(HIPAA). Those regulations require a signed subject authorization informing the subject 
of the following: 
• What protected health information (PHI) will be collected from subjects in this 
study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI. 
In the event that a subject revokes authorization to collect or use PHI, the investigator, 
by regulation, retains the ability to use all information collected prior to the revocation of 
subject authorization. For subjects that have revoked authorization to collect or use 


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PHI, attempts should be made to obtain permission to collect at least vital status (i.e. 
that the subject is alive) at the end of their scheduled study period. 
 
All participating parents / guardians will sign a HIPAA form for this study. 
 
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial. 
Source data are contained in source documents. Examples of these original 
documents, and data records include: hospital records, clinical and office charts, 
laboratory notes, memoranda, subjects’ diaries or evaluation checklists, pharmacy 
dispensing records, recorded data from automated instruments, copies or transcriptions 
certified after verification as being accurate and complete, microfiches, photographic 
negatives, microfilm or magnetic media, x-rays, subject files, and records kept at the 
pharmacy, at the laboratories, and at medico-technical departments involved in the 
clinical trial. 
 
Data generated by the methods described in the protocol will be recorded in the 
subjects' medical records and/or study progress notes. Data may be transcribed legibly 
on CRFs supplied for each subject or directly inputted into an electronic system or any 
combination thereof. 
 
9.3 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study. 
All data requested on the CRF must be recorded. All missing data must be explained. 
If a space on the CRF is left blank because the procedure was not done or the question 
was not asked, write “N/D”. If the item is not applicable to the individual case, write 
“N/A”. All entries should be printed legibly in black ink. If any entry error has been 
made, to correct such an error, draw a single straight line through the incorrect entry 
and enter the correct data above it. All such changes must be initialed and dated. DO 
NOT ERASE OR WHITE OUT ERRORS. For clarification of illegible or uncertain 
entries, print the clarification above the item, then initial and date it. 
 
Source documents will serve as case report forms for all test article and behavioral 
survey forms completed by the subjects’ parent/guardian and for all of the standardized 
FASD and neurodevelopment tests implemented as a part of this research. 
 
9.4 Records Retention 
It is the investigator’s responsibility to retain study essential documents for at least 2 
years after the last approval of a marketing application in their country and until there 
are no pending or contemplated marketing applications in their country or at least 2 
years have elapsed since the formal discontinuation of clinical development of the 
investigational product. These documents should be retained for a longer period if 
required by an agreement with the sponsor. In such an instance, it is the responsibility 
of the sponsor to inform the investigator/institution as to when these documents no 
longer need to be retained. 


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10. Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
This study will be monitored according to FDA/GCP guidelines by the University’s 
Clinical and Translational Science Institute monitoring service. The investigator will 
allocate adequate time for such monitoring activities. The Investigator will also ensure 
that the monitor or other compliance or quality assurance reviewer is given access to all 
the above noted study-related documents and study related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.), and has adequate space to conduct the monitoring visit. 
 
10.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, 
the sponsor, government regulatory bodies, and University compliance and quality 
assurance groups of all study related documents (e.g. source documents, regulatory 
documents, data collection instruments, study data etc.). The investigator will ensure 
the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection 
by government regulatory authorities and applicable University compliance and quality 
assurance offices. 
 
11.0 Ethical Considerations 
This study is to be conducted according to US and international standards of Good 
Clinical Practice (FDA Title 21 part 312 and International Conference on Harmonization 
guidelines), applicable government regulations and Institutional research policies and 
procedures. 
 
This protocol and any amendments will be submitted to a properly constituted 
independent Institutional Review Board (IRB), in agreement with local legal 
prescriptions, for formal approval of the study conduct. The decision of the IRB 
concerning the conduct of the study will be made in writing to the investigator and a 
copy of this decision will be provided to the sponsor before commencement of this 
study. The investigator should provide a list of IRB members and their affiliate to the 
sponsor. 
 
All subjects for this study will be provided a consent form describing this study and 
providing sufficient information for subjects to make an informed decision about their 
participation in this study. The consent form will be submitted with the protocol for 
review and approval by the IRB for the study. The formal consent of a subject, using 
the IRB-approved consent form, must be obtained before that subject is submitted to 
any study procedure. This consent form must be signed by the subject or legally 
acceptable surrogate, and the investigator-designated research professional obtaining 
the consent. 


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12. Study Finances 
12.1 Funding Source 
The study is funded by grants from the National Institutes on Alcohol Abuse and 
Alcoholism / National Institutes of Health (1R01AA024123 and 3R01AA024123- 
05S1). 
 
12.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, 
royalties, or financial gain greater than the minimum allowable by their institution, etc.) 
must refer to the Regents Policies on Individual Conflict of Interest Policy or Institutional 
Conflict of Interest Policy. These policies require University Faculty and staff to report 
external professional activities and business and significant financial interests related to 
his or her University activities by submitting a REPA (Report of External Professional 
Activities) at least once per year. Faculty and staff should also file a REPA when 
substantial changes in business or financial interests occur, when an activity that 
presents a potential conflict of interest is anticipated, or when submitting an application 
for research support or technology transfer, submitting research protocols to the IRB, or 
receiving financial contributions. All University of Minnesota investigators will follow the 
University conflict of interest policy. 
 
12.3 Subject Stipends or Payments 
 
Participant families will receive payment for participation. Payment will be made using a 
pre-paid debit card called Greenphire ClinCard. It works like a bank debit card. We will 
give participants a debit card and the money will be added to the card after each 
completed visit. Because the participants are children, the payment will be provided to the 
parent on behalf of the child. Payments will be made following each visit. Payment will be 
based on procedures attempted and/or completed as follows: 
 
Visits 1 & 11: $100 each 
Visits 2, 3, 4, 5, 6, 7, 9, 10, 12: $10 each 
Visit 8: $75 
 
The maximum payment per participant will be $365 over the course of 10 months. 
 
13. Publication Plan 
Neither the complete nor any part of the results of the study carried out under this 
protocol, nor any of the information provided by the sponsor for the purposes of 
performing the study, will be published or passed on to any third party without the 
consent of the study sponsor. Any investigator involved with this study is obligated to 
provide the sponsor with complete test results and all data derived from the study. 
 
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Dehaene, P., Hanson, J. W., & Graham, J. M., Jr. (1997). Incidence of fetal alcohol 
syndrome and prevalence of alcohol-related neurodevelopmental disorder. 
Teratology, 56(5), 317-326. 
Sanders, L. M., & Zeisel, S. H. (2007). Choline: Dietary Requirements and Role in Brain 
Development. Nutr Today, 42(4), 181-186. 


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This material is the property of the University of Minnesota. Do not disclose or use except as authorized in writing by the study sponsor 
 
 
 
 
Shaw, G. M., Carmichael, S. L., Yang, W., Selvin, S., & Schaffer, D. M. (2004). 
Periconceptional dietary intake of choline and betaine and neural tube defects in 
offspring. Am J Epidemiol, 160(2), 102-109. 
Sheard, N. F., & Zeisel, S. H. (1986). An in vitro study of choline uptake by intestine 
from neonatal and adult rats. Pediatr Res, 20(8), 768-772. 
Smithells, R. W., Sheppard, S., & Schorah, C. J. (1976). Vitamin dificiencies and neural 
tube defects. Arch Dis Child, 51(12), 944-950. 
Sokol, R. J., Miller, S. I., & Reed, G. (1980). Alcohol abuse during pregnancy: an 
epidemiologic study. Alcohol Clin Exp Res, 4(2), 135-145. 
Sowell, E. R., Mattson, S. N., Thompson, P. M., Jernigan, T. L., Riley, E. P., & Toga, A. 
W. (2001). Mapping callosal morphology and cognitive correlates: Effects of heavy 
prenatal alcohol exposure. Neurology, 57(2), 235-244. 
Tang, W. H., Wang, Z., Levison, B. S., Koeth, R. A., Britt, E. B., Fu, X., et al. (2013). 
Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N 
Engl J Med, 368(17), 1575-1584. 
Thomas, J. D., Biane, J. S., O'Bryan, K. A., O'Neill, T. M., & Dominguez, H. D. (2007). 
Choline supplementation following third-trimester-equivalent alcohol exposure 
attenuates behavioral alterations in rats. Behav Neurosci, 121(1), 120-130. 
Thomas, J. D., Garrison, M., & O'Neill, T. M. (2004). Perinatal choline supplementation 
attenuates behavioral alterations associated with neonatal alcohol exposure in rats. 
Neurotoxicol Teratol, 26(1), 35-45. 
Thomas, J. D., La Fiette, M. H., Quinn, V. R., & Riley, E. P. (2000). Neonatal choline 
supplementation ameliorates the effects of prenatal alcohol exposure on a 
discrimination learning task in rats. Neurotoxicol Teratol, 22(5), 703-711. 
Utsunomiya, H., Takano, K., Okazaki, M., & Mitsudome, A. (1999). Development of the 
temporal lobe in infants and children: analysis by MR-based volumetry. AJNR Am J 
Neuroradiol, 20(4), 717-723. 
Wagner, A. F., & Hunt, P. S. (2006). Impaired trace fear conditioning following neonatal 
ethanol: reversal by choline. Behav Neurosci, 120(2), 482-487. 
Wang, Z., Klipfell, E., Bennett, B. J., Koeth, R., Levison, B. S., Dugar, B., et al. (2011). 
Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. 
Nature, 472 (7341), 57-63. 
Ward, B. C., Kolodny, N. H., Nag, N., & Berger-Sweeney, J. E. (2009). Neurochemical 
changes in a mouse model of Rett syndrome: changes over time and in response to 
perinatal choline nutritional supplementation. J Neurochem, 108(2), 361-371. 
Warren, K., Floyd, L., Calhoun, F., Stone, D., Bertrand, J., & Streissguth, A. (2004). 
Consensus statement on FASD. Washington D.C.: National Organization on Fetal 
Alcohol Syndrome. 
Williams, C. L. (2008). Food for thought: brain, genes, and nutrition. Brain Res, 1237, 1- 
4. 
Williams, C. L., Meck, W. H., Heyer, D. D., & Loy, R. (1998). Hypertrophy of basal 
forebrain neurons and enhanced visuospatial memory in perinatally choline- 
supplemented rats. Brain Res, 794(2), 225-238. 
Wong-Goodrich, S. J., Glenn, M. J., Mellott, T. J., Blusztajn, J. K., Meck, W. H., & 
Williams, C. L. (2008). Spatial memory and hippocampal plasticity are differentially 


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sensitive to the availability of choline in adulthood as a function of choline supply in 
utero. Brain Res, 1237, 153-166. 
Wozniak, J. R., Fuglestad, A. J., Eckerle, J. K., Kroupina, M. G., Miller, N. C., Boys, C. 
J., Brearley, A. M., Fink, B. A., Hoecker, H. L., Zeisel, S. H., & Georgieff, M. K. 
(2013). Choline supplementation in children with Fetal Alcohol Spectrum Disorders 
(FASD) has high feasibility and tolerability. Nutrition Research, 33(11), 897-904. 
Wozniak, J. R., Fuglestad, A. J., Eckerle, J. K., Fink, B. A., Hoecker, H. L., Boys, C. J., 
Radke, J., Kroupina, M. G., Miller, N. C., Brearley, A. M., Zeisel, S. H., & Georgieff, 
M. K., (under review). Choline Supplementation in children with Fetal Alcohol 
Spectrum Disorders (FASD): A randomized, double-blind, placebo-controlled trial, 
American Journal of Clinical Nutrition. 
Xu, X., Gammon, M. D., Zeisel, S. H., Lee, Y. L., Wetmur, J. G., Teitelbaum, S. L., 
Bradshaw, P. T., Neugut, A. I., Santella, R. M., & Chen, J. (2008). Choline 
metabolism and risk of breast cancer in a population-based study. Faseb J, 22(6), 
2045-2052. 
Yen, C. L., Mar, M. H., Meeker, R. B., Fernandes, A., & Zeisel, S. H. (2001). Choline 
deficiency induces apoptosis in primary cultures of fetal neurons. Faseb J, 15(10), 
1704-1710. 
Zeisel, S. H. (1992). Choline: an important nutrient in brain development, liver function 
and carcinogenesis. J Am Coll Nutr, 11(5), 473-481. 
Zeisel, S. H. (1997). Choline: essential for brain development and function. Adv Pediatr, 
44, 263-295. 
Zeisel, S. H. (2000). Choline: needed for normal development of memory. J Am Coll 
Nutr, 19(5 Suppl), 528S-531S. 
Zeisel, S. H. (2004). Nutritional importance of choline for brain development. J Am Coll 
Nutr, 23(6 Suppl), 621S-626S. 
Zeisel, S. H. (2006). The fetal origins of memory: the role of dietary choline in optimal 
brain development. J Pediatr, 149(5 Suppl), S131-136. 
Zeisel, S. H. (2007). Gene response elements, genetic polymorphisms and epigenetics 
influence the human dietary requirement for choline. IUBMB Life, 59(6), 380-387. 
Zeisel, S. H. (2008). Genetic polymorphisms in methyl-group metabolism and 
epigenetics: lessons from humans and mouse models. Brain Res, 1237, 5-11. 
Zeisel, S. H. (2009a). Importance of methyl donors during reproduction. Am J Clin Nutr, 
89(2), 673S-677S. 
Zeisel, S. H. (2009b). Is maternal diet supplementation beneficial? Optimal development 
of infant depends on mother's diet. Am J Clin Nutr, 89(2), 685S-687S. 
Zeisel, S. H., Albright, C. D., Shin, O. H., Mar, M. H., Salganik, R. I., & da Costa, K. A. 
(1997). Choline deficiency selects for resistance to p53-independent apoptosis and 
causes tumorigenic transformation of rat hepatocytes. Carcinogenesis, 18(4), 731- 
738. 
Zeisel, S. H., Da Costa, K. A., Franklin, P. D., Alexander, E. A., Lamont, J. T., Sheard, 
N. F., & Beiser, A. (1991). Choline, an essential nutrient for humans. Faseb J, 5(7), 
2093-2098. 
Zeisel, S. H., Mar, M. H., Howe, J. C., & Holden, J. M. (2003). Concentrations of 
choline-containing compounds and betaine in common foods. J Nutr, 133(5), 1302- 
1307. 


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Zeisel, S. H., & Niculescu, M. D. (2006). Perinatal choline influences brain structure and 
function. Nutr Rev, 64(4), 197-203. 
 
15. Attachments 
1. Study Schedule of Events 


Clinical Research Protocol Template 
Page 1 
 
Version June 30, 2015 
 
 
 
Visit Type 
Clinic 
Phone 
Phone 
Phone 
Phone 
Phone 
Phone 
Clinic 
Phone 
Phone 
Clinic 
Phone 
Visit Title 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 
Visit 10 
Visit 11 
Visit 12 
Time Point 
Baseline 
Day 14 
Day 28 
Month 2 
Month 3 
Month 4 
Month 5 
Month 6 
Month 7 
Month 8 
Month 9 
Month 
10 
Study Visit Window 
n/a 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
+ or - 14 
days 
Consent 
X 
Medical/developmental 
history 
X 
Physical examination 
X 
X 
X 
X 
Height 
X 
X 
X 
X 
Weight 
X 
X 
X 
X 
Head circumference 
X 
X 
X 
Blood pressure 
X 
X 
X 
X 
Blood collection for choline 
level 
X 
X 
X 
DNA sample from same 
blood sample 
X 
Astley and Clarren's FASD 
diagnosis with photograph 
of the child's face 
X 
Stanford Binet 
X 
X 
Elicited Imitation with video 
X 
X 
X 
MEFS Early Childhood 
Version 
X 
X 
X 
NIH Toolbox Flanker Task 
X 
X 
X 
Beery VMI Test 
X 
Scales of Indep. Behavior 
X 
Child Behavior Checklist 
X 
X 
X 
24 hour diet Interview 
X 
X 
X 
Child Eating Behavior 
Questionnaire 
X 
Randomization 
X 


Page 2 
 
 
 
Parent/guardian training in 
proper storage & 
administration of test article 
X 
Dispense test agent 
(choline/placebo) 
X from 
thereon 
as  
needed 
Record concomitant 
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Record adverse events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Compliance check (packet 
count) 
X 
X 
Record parent-reported 
problems with 
administration of test article 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CONFIDENTIAL 
This material is the property of the University of Minnesota. Do not disclose or use except as authorized in writing by the study sponsor 

ICF_000.pdf: 
UNIVERSITY OF MINNESOTA  
National Institute of Health / Protocol Number 1R01AA024123 
Page 1 of 11 
Initials __________ Date __________ 
Jeffrey R. Wozniak, PhD 
Advarra IRB Approved Version 10 Jan 2020 
INFORMATION AND CONSENT FORM 
For Parents/Guardians of New Participants 
Sponsor / Study Title: 
National Institute of Health / “Choline 
Supplementation As A Neurodevelopmental 
Intervention in Fetal Alcohol Spectrum Disorders” 
Protocol Number: 
1R01AA024123 
Principal Investigator: 
Jeffrey R. Wozniak, PhD 
Telephone: 
(612) 273-9741
(612) 273-6212 (24-Hours)
Address: 
Psychiatry Department - F282/2A West 
2450 Riverside Ave. 
Minneapolis, MN  55454 
The study in investigator wants to know if you and your child would like to be part of a research study. If 
you have any questions about or do not understand something in this form, you should ask the study 
investigator or study staff.  You should also discuss your and your child's participation with anyone you 
choose in order to better understand this study and your and your child's options.  Participating in a 
research study is not the same as getting regular medical care.  The purpose of regular medical care is 
to improve health.  The purpose of a research study is to gather information. 
WHAT IS THIS STUDY ABOUT? 
Researchers want to find out more about a nutrient called choline. For purposes of this study, choline is 
regulated and monitored as an "investigational drug" by the U.S. Food and Drug Administration (FDA) 
because it is being tested as a potential treatment for Fetal Alcohol Spectrum Disorder (FASD), and is 
not approved in the United States by the FDA. The main purpose of this study is to see whether choline 
can help cognitive development (thinking / memory) in children with FASD.   
The study will also compare choline with a placebo to see if taking choline is better than taking a 
placebo.  The placebo in this study looks and tastes like choline but has no choline in it.  About 60 
children with FASD will be in this study. Throughout this form, the term "study drug" refers to choline 
and placebo. 
NCT02735473


National Institute of Health / Protocol Number 1R01AA024123 
Page 2 of 11  
 
 
Initials __________ Date __________ 
 
Jeffrey R. Wozniak, PhD 
 
Advarra IRB Approved Version 10 Jan 2020 
 
HOW DOES CHOLINE WORK? 
Choline is a nutrient found in many foods that your child eats.  Choline is grouped within the vitamin B 
complex. The Institute of Medicine (IOM, a non-profit division of the National Academies of Sciences, 
Engineering and Medicine) recommends that children ages 1-3 eat 200 mg. of choline per day while 
those ages 4-8 eat 250 mg per day.  The IOM's recommended upper tolerable intake level is 1000 mg 
per day for children aged 1-8 years. 
Prenatal alcohol exposure can damage the brain during pregnancy, and children with prenatal alcohol 
exposure can have learning and behavioral problems as a result (including difficulties with attention, 
memory, problem-solving, and behavioral control).  Recent studies in animals suggest that nutrients, 
like choline, may be helpful to brain development - even after the initial damage from alcohol has 
occurred.  These studies also suggest that choline may contribute to long-term improvements in 
learning.  Because this is one of the first studies of its kind, we do not yet know whether choline will 
actually help children with FASD. 
 
IS THERE ANYTHING ELSE MY CHILD CAN DO FOR HIS/HER FASD? 
If you decide not to allow your child to participate in this study, your child's normal clinical care will 
continue.  Alternatives to participating in this study could involve behavioral interventions and/or special 
education services for your child.  There are no specific biological interventions for FASD. 
You should discuss your child's alternatives to participating in this research with the study investigator 
or study staff.  In addition, you may discuss your options with your child's regular health care provider. 
 
WHAT IS MY ALTERNATIVE TO BEING IN THE STUDY? 
Your participation in this study does not involve treatment for any condition. Your alternative is to not 
participate in the study. 
 
WHO IS PAYING FOR THIS STUDY? 
The National Institutes of Health (NIH), the sponsor of the study, is paying for this study.  
 
WILL IT COST ANYTHING TO BE IN THIS STUDY? 
The sponsor will cover all costs associated with this study. 
 
HOW LONG WILL MY CHILD AND I BE IN THE STUDY? 
If you decide to be in this study and to allow your child to be in the study, and the study investigator 
says you and your child can be in the study, your and your child's participation will last 10 months (9 
months in the study and one follow-up call at 10 months).   
• 
If you decide to be in this study and to allow your child to be in the study, the screening will be 
completed during the first visit.  
• 
If you and your child continue your participation past the first visit, your child will begin the study 
drug dosing period, as described in this form.  Your child may continue to receive study drug for 


National Institute of Health / Protocol Number 1R01AA024123 
Page 3 of 11  
 
 
Initials __________ Date __________ 
 
Jeffrey R. Wozniak, PhD 
 
Advarra IRB Approved Version 10 Jan 2020 
 
up to 9 months unless you and/or the study investigator decide to stop your child's study drug 
dosing.  
• 
If study drug dosing is stopped, you and your child may continue your study participation by 
taking part in the study's follow-up period (described in this form). 
You and your child will visit the study center 3 times to have the procedures and tests described in this 
form.  Ask the study investigator or study staff about your and your child's study visit schedule.  
 
WHAT WILL HAPPEN DURING THIS STUDY? 
The study investigator or study staff will give you a powdered drink mix containing choline or placebo. 
You will mix it with water and your child will drink it once per day for nine months.  
Your child will be assigned by chance (like flipping a coin) to one of the following study groups: 
• 
Group 1:  choline bitartrate 19 mg. per kg. (weight based dosing) 
• 
Group 2:  placebo (a drink mix that looks and tastes like choline bitartrate but has no choline in 
it). 
Your child has an equal chance of being in either of the study groups.  Your child may receive a 
placebo.  Neither you nor the study investigator or study staff will be able to pick which study group 
your child is in.  You will not know and the study investigator and study staff will not know which study 
group your child is in.  The study investigator or study staff can find out if it is necessary to know for 
your child's health.  If this happens and the study investigator decides that it is best to withdraw your 
child from the study, you will be told which group your child was in. On the other hand, if the study 
investigator decides that your child can safely continue in the study, you will not be told which group 
your child is in. 
While you and your child are in the study, you must: 
• 
Follow the instructions you are given. 
• 
Come to the study center for all visits with the study investigator or study staff. 
• 
Tell the study investigator or study staff about any changes in your child's health or how they 
feel. 
• 
Tell the study investigator or study staff if you want to stop being in the study at any time, or if 
you want your child to stop being in the study at any time. 
 
What happens when we come for study visits? 
After you sign this form, the study investigator or study staff will do the things listed below when you 
and your child come in for study visits.  If you would like more information about which tests and 
procedures will be done at each study visit, ask the study investigator or study staff.   
• 
Demographic Questions:  Ask you to give personal information, such as your name, your 
child's name, date of birth, race, telephone numbers, etc. 
• 
Health and Medication Questions:  Ask you to answer questions about your child's health, 
medical history, and the medications he or she takes. 
• 
Behavioral Questionnaires:  Ask you to fill out questionnaires about your child's emotional, 
behavioral, and cognitive functioning. 
• 
Dietary Interview:  You will be asked about your child's diet, covering a 24-hour period, on 
three occasions. 


National Institute of Health / Protocol Number 1R01AA024123 
Page 4 of 11  
 
 
Initials __________ Date __________ 
 
Jeffrey R. Wozniak, PhD 
 
Advarra IRB Approved Version 10 Jan 2020 
 
• 
Physical Exam:  Your child will have a brief facial examination including measurements. The 
study investigator will also look at your child's hands and will ask basic health questions. 
• 
Photograph and Video: Your child's face will be photographed to allow for detailed 
measurements to be taken. Your child will be video-recorded while completing one of the 
memory tasks to make it easier to score the test afterward.  We will not share or release the 
photograph or the video recording outside of the study. The photograph and video will become 
part of the study record. You will read more about the photography and video later in this form. 
• 
Vital Signs:  Check your child's blood pressure by putting a band around the arm (this will 
squeeze the arm for about a minute). 
• 
Height, Weight:  Measure how tall your child is and check how much he or she weighs. 
• 
Blood Testing:  Take some blood to do laboratory tests: 
o Checking levels of certain substances in your child's body to determine how much 
change occurs with choline supplementation 
o DNA extraction from the same blood sample to look at genetic differences related to how 
much choline different children's bodies can make on their own. You will read more 
about this DNA testing later in this form. 
• 
Cognitive testing:  Your child will have several paper and pencil and interactive assessments 
including measures of brain function, thinking, memory, and behavior.  Your child will be 
observed in structured play activities and will be asked to perform tasks such as manipulating 
toys, answering questions, and pointing. 
• 
Study Diary:  Give you a calendar log sheet for recording days on which your child did and did 
not take the full amount of the study drug. 
• 
Study Drug:  Give you a supply of study drug.  Ask you to bring back all unused study drug to 
each visit. 
What else will happen during this study? 
• 
Phone visits:  Between visits at the study center, we will call you approximately once per 
month to check for potential side effects and see if you need any assistance with the study. 
• 
Blood samples:  After the blood tests described in this form have been completed, your child's 
samples will be destroyed. 
 
WHY WILL MY CHILD BE PHOTOGRAPHED AND VIDEO-RECORDED? HOW WILL THESE BE 
USED FOR THE STUDY? 
The study investigator or study staff will take a photograph of your child's face.  The purpose is to allow 
for computerized measurement of facial features such as eye width measurement and lip flatness. 
These facial characteristics can sometimes be affected by prenatal alcohol exposure.  The study 
investigator or study staff will also video-record your child during the performance of one of the memory 
tasks.  This is necessary in order to allow for very detailed scoring of your child's performance 
afterward.  You do not have to let the study investigator or study staff take the photo and video of your 
child if you don't want to. However, your child cannot be part of the study without the facial photograph 
and the video recording of the memory task.  You can ask the study investigator or study staff 
questions about it before you decide if you want to let them take the photo and video of your child. 
The photo and video will show your child's full face.  It is possible that people who see the photograph 
and/or video will recognize your child. 
Your child's photograph and video recording will be identified by a number only (not a name) and will be 
stored on a secure file server and not viewed by anyone who is not authorized to see it or published or 


National Institute of Health / Protocol Number 1R01AA024123 
Page 5 of 11  
 
 
Initials __________ Date __________ 
 
Jeffrey R. Wozniak, PhD 
 
Advarra IRB Approved Version 10 Jan 2020 
 
used at scientific meetings.  You will not receive copies of the photograph or video and you will not be 
paid for the photograph or video. 
 
HOW WILL MY CHILD'S DNA (GENES) BE USED? 
The study investigators will examine your child's DNA (genes) in order to better understand the 
relationship between individual differences and response to choline supplementation. DNA is in your 
child's cells, and it is what makes him/her different from anyone else. Some DNA controls things like 
the color of your child's hair or eyes. DNA might make your child more likely to get certain diseases or 
affect whether a drug helps your child and/or gives them side effects. 
We believe that some children may respond better to choline depending on certain differences in 
specific genes.  These differences are called single nucleotide polymorphisms (SNPs). Your child's 
DNA will be extracted from the blood sample collected at the first visit.  His or her DNA sample will be 
stored until it can be analyzed in bulk with other samples. This will occur during the course of the 
current study.  Once your child's DNA sample has been analyzed for these SNPs, it will be destroyed.  
There is no physical risk to the genetic portion of the study - other than the risks associated with the 
blood draw (described elsewhere in this form).  The genetics study is a required part of the main 
study.   
You and your child will not receive any results from this genetic test and the test results will not be put 
in your child's regular medical records. The test will not cost you anything. This DNA analysis will not 
benefit you or your child, but will help researchers understand choline as a potential treatment in FASD. 
The risks to you and your family from genetic research are low. Your child's samples will be identified 
only with his/her study code number. If you change your mind later about allowing your child to be in 
the study, and the sample has not yet been analyzed, tell the study investigator or study staff and they 
will destroy your child's DNA sample. 
There are laws to protect privacy and to prohibit the misuse of your child's genetic information.  
However, it is important to know that there is still a risk someone could get access to the information we 
have stored about your child, including information about a family member (for example, revealing that 
your child or a blood relative carry a genetic disease). 
 
WILL BEING IN THIS STUDY HELP ME OR MY CHILD? 
Choline may help your child's symptoms from prenatal alcohol exposure, but there is no guarantee that 
being in this study will help your child.  Your child's FASD might not get better while he/she is in this 
study.  Your child may get placebo, which looks and tastes like choline bitartrate, but has no choline in 
it.  Information from this study might help researchers to better understand FASD or come up with new 
tests or medications to help others in the future.  
 
WHAT ARE THE RISKS TO ME AND MY CHILD IF WE ARE IN THIS STUDY? 
What are the risks of taking choline? 
At very high doses (15 to 30 times higher than the dose we will be using), some people have reported: 
• 
Hypotension or lowered blood pressure  
• 
Gastrointestinal symptoms (upset stomach, stomach pain, loss of appetite) 


National Institute of Health / Protocol Number 1R01AA024123 
Page 6 of 11  
 
 
Initials __________ Date __________ 
 
Jeffrey R. Wozniak, PhD 
 
Advarra IRB Approved Version 10 Jan 2020 
 
• 
One side effect we did observe in our previous study at a fixed dose of 500 mg. per day was 
"fishy" body odor (noticeable in urine and in sweat).  The dose to be used in this study is 
generally lower than in the previous study.  The dose will be adjusted to your child's weight at 
the first visit.  If your child is randomly assigned to choline, he or she will receive a dose of 19 
mg. of choline per kg body weight.  For example, if your child has a weight of 15 kg at the first 
visit, the daily dose will be 285 mg.  The maximum daily dose amount to be used in this study 
(500 mg.) is well within the range that could be achieved by most children through dietary 
choices and the maximum dosage to be used is half of the "upper tolerable intake level" for 
children in this age range (1000 mg.). 
Please tell the study investigator or study staff right away if your child has any side effects.  Please tell 
them if your child has other problems with health or the way he or she feels during the study, whether 
or not you think these problems are related to the study drug. 
It is possible that choline may change how your child's regular medications, vaccines, vitamins or 
supplements work.  It is important that you tell the study investigator about any medications, vitamins or 
supplements before you give them to your child during the study, or if your child will receive a vaccine 
during the study. 
 
Could my child have an allergic reaction? 
Sometimes people have allergic reactions to drugs or product ingredients.  If your child has a very bad 
allergic reaction, he/she could die. Some things that happen during an allergic reaction that could be a 
sign or symptom of a life-threatening allergic reaction (anaphylaxis) are: 
• 
a rash  
• 
a fast pulse 
• 
sweating 
• 
a feeling of dread 
• 
swelling around the eyes and mouth 
• 
swelling of the throat 
• 
wheezing 
• 
having a hard time breathing 
• 
a sudden drop in blood pressure (making your child feel dizzy or lightheaded) 
• 
inability to breathe without assistance 
You should get medical help and contact the study investigator or study staff if your child has any of 
these or any other side effects during the study. 
 
What if my child is receiving placebo instead of choline during the study? 
Some children in the study will get placebo instead of choline.  In this study, the placebo is a drink mix 
that looks and tastes like choline but has no choline in it.  Please ask the study investigator or study 
staff if you have any questions about placebo. 
 
What are the risks of giving blood for this study? 
The study investigator or study staff will take your child's blood by sticking a needle in the arm.  Some 
problems your child might have from this are: 


National Institute of Health / Protocol Number 1R01AA024123 
Page 7 of 11  
 
 
Initials __________ Date __________ 
 
Jeffrey R. Wozniak, PhD 
 
Advarra IRB Approved Version 10 Jan 2020 
 
• 
pain 
• 
bruising 
• 
dizziness 
• 
infection 
 
What are the other risks? 
Filling out the questionnaires and answering the study investigator or study staff's questions could lead 
you to feel uncomfortable or upset.  Please tell the study investigator or study staff if you feel 
uncomfortable or upset while filling out a questionnaire or answering questions.  You have the right to 
refuse to answer any questions.   
There is a risk of loss of confidentiality of your and your child's information.  You will read more about 
the protection of your and your child's information later in this form.  Please ask the study investigator 
or study staff if you would like to know more about how your and your child's information will be 
protected while you and your child are in this study. 
 
COULD MY CHILD HAVE ANY OTHER HEALTH PROBLEMS IF HE/SHE IS IN THIS STUDY? 
It is possible that your child could have problems or side effects of choline that nobody knows about 
yet, which include his/her health getting worse.  Ask the study investigator if you have questions about 
the signs or symptoms of any side effects that you read about in this consent form. 
 
WILL I RECEIVE ANY NEW INFORMATION DURING THE STUDY? 
If the study investigator or study staff learns any new information that might change your mind about 
continuing in the study, or allowing your child to continue in the study, the study investigator or study 
staff will tell you about it. 
 
WHAT IF MY CHILD GETS HURT OR SICK WHILE IN THIS STUDY? 
In the event that this research activity results in an injury, treatment will be available, including first aid, 
emergency treatment and follow-up care as needed.  Care for such injuries will be billed in the ordinary 
manner, to you or your child's insurance company.  Be aware that your child's health care payer/insurer 
might not cover the costs of study-related injuries or illnesses.  The sponsor has no plans to provide 
any payment for medical treatment.  If you think that your child has suffered a research related injury, 
let the study investigator or study staff know right away. You do not give up any of your or your child's 
legal rights by signing this form. 
 
WILL MY CHILD OR I RECEIVE PAYMENT? 
Payment will be made using a pre-paid debit card called Greenphire ClinCard. It works like a bank debit 
card. We will give you a debit card; each time you are paid for a visit, money will be added to the card. 
You may use this card at any store that accepts MasterCard or you can use a bank machine to get 
cash. However, there may be fees for cash withdrawals (ATM use) and inactivity (no use for 3 months). 
We will give you the ClinCard Frequently Asked Questions information sheet that answers common 
questions about the debit card. You will also receive letters with additional information on how you can 


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Advarra IRB Approved Version 10 Jan 2020 
 
use this card and who to call if you have any questions. Be sure to read these letters, including the 
cardholder agreement, for details about fees.  
The card is administered by an outside company. The company, Greenphire, will be given your name. 
They will use this information only as part of the payment system. Your information will not be used for 
any other purposes and will not be given or sold to any other company. Greenphire will not receive any 
information about your health status or the study in which you are participating. You will have the option 
to receive updates related to appointment reminders and payment reminders and updates via text 
message and email message (Standard text messaging rates will apply). You will have the opportunity 
to opt-in to receive these messages; you are not required to provide your phone number or email to be 
in the study or use a ClinCard. If you choose to receive messages but later decide that you want to stop 
the messages, you can opt-out. Any demographic information collected and provided to Greenphire is 
stored in a secure fashion and will be kept confidential, except as required by law. 
____Yes, I would like to receive notifications from Greenphire 
Email Address:  ______________________________ 
Phone Number: ______________________________ 
____No, I do not want to receive notifications from Greenphire 
Payment you receive as compensation for research is considered taxable income. If payment to an 
individual exceeds $600 in any one calendar year, the University of Minnesota is required to report this 
to the Internal Revenue Service (IRS). Research payments to study participants exceeding $600.00 
during any calendar year will result in a FORM 1099 (Miscellaneous Income) being issued to you and a 
copy sent to the IRS. 
You will be paid for each study visit you complete. The payment schedule is as follows: 
Visits 1 & 11 (In-person): $100 each 
Visits 2, 3, 4, 5, 6, 7, 9, 10, 12 (Phone visits): $10 each 
Visit 8 (In-person): $75 
If you complete all of the study visits you will receive a maximum payment of $365. 
If you and your child travel to the study center, you will also be reimbursed up to $0.575 per mile. If you 
live more than 2 hours from Minneapolis, you may also be eligible for hotel reimbursement. You may 
also be reimbursed for parking costs at the study center (approximately $5-10 per visit). Please ask the 
study investigator or study staff if you have questions about any reimbursements you and your child 
may be eligible for during the study. 
 
DO MY CHILD OR I HAVE TO BE IN THIS STUDY? 
Your and your child's participation in this study is voluntary.  You can decide not to be in the study, and 
not to allow your child to be in the study, and you can change your mind at any time.  There will be no 
penalty to you or your child, and you and your child won't lose any benefits except for benefits having to 
do with the study.  If you want to stop being in the study, or if you want to take your child out of the 
study, tell the study investigator or study staff. 
The study investigator or study staff or sponsor can remove you or your child from the study at any 
time, even if you want to stay in the study or you want your child to stay in the study.  This could 
happen if:  


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• 
The study investigator or study staff believes it is best for you or your child to stop being in the 
study. 
• 
You do not follow directions about the study. 
• 
The sponsor stops the study for any reason. 
If you or your child stop being in the study early, the study investigator or study staff may ask you some 
questions about being in the study.  The study investigator or study staff may ask you and your child to 
participate in some procedures or tests to help you leave the study safely and/or to collect more 
information for the study.  If you or your child leaves the study, the study investigator and study staff will 
still be able to use your information and your child's information that they have already collected.  The 
study investigator or study staff will try to complete the monthly telephone calls with you through the 10 
month point, even if you or your child discontinue earlier. 
If you change your mind about allowing your child to be in the study later, tell the study investigator or 
study staff and they will destroy your child's blood samples if they are still available. 
 
HOW WILL MY AND MY CHILD'S INFORMATION BE KEPT CONFIDENTIAL? 
Your identity and your child's identity will be protected as required by law and according to any policies 
the study center or sponsor may have.  Be aware that your and your child's study records (which 
include your child's medical records, your signed consent form, and other information) will be shared as 
needed for the study.  For example, the study investigator and the study staff members, the University 
of Minnesota, University of Minnesota Physicians, Fairview Health Services, the U.S. Food and Drug 
Administration (FDA), the Office for Human Research Protections (OHRP), the National Institutes of 
Health, and Advarra IRB may look at your and your child's study and medical records. 
Your documents and your child's documents and samples will not be labeled with a name or other 
directly identifying information.  The documents and samples will have a code instead.  The list that 
matches the code with your name and your child's name will be stored separately from your documents 
and your child's documents and samples. 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by U.S. 
Law.  This Web site will not include information that can identify you.  At most, the Web site will include 
a summary of the results.  You can search this Web site at any time. 
 
WHOM TO CONTACT ABOUT THIS STUDY 
During the study, if you experience any medical problems, suffer a research-related injury, or have 
questions, concerns or complaints about the study, please contact the study investigator at the 
telephone number listed on the first page of this consent document. If you seek emergency care, or 
hospitalization is required, alert the treating physician that you are participating in this research study.  
An institutional review board (IRB) is an independent committee established to help protect the rights of 
research participants. If you have any questions about your rights as a research participant, and/or 
concerns or complaints regarding this research study, contact: 
• 
By mail: 
Study Participant Adviser 
Advarra IRB 


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6940 Columbia Gateway Drive, Suite 110 
Columbia, MD 21046 
• 
or call toll free:    877-992-4724 
• 
or by email:          adviser@advarra.com 
Please reference the following number when contacting the Study Participant Adviser: Pro00038437. 
 
 


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Advarra IRB Approved Version 10 Jan 2020 
 
 
CONSENT 
I am the parent/guardian of the child being asked to participate in this study.  I have read this form, and 
I have been able to ask questions about this study.  The study investigator or study staff has talked with 
me about this study.  They have answered all my questions.  I voluntarily agree to be in this study and 
allow my child to be in this study.  By signing this form, I do not give up any of my or my child's legal 
rights.  I will get a signed copy of this consent form. 
 
 
 
Printed Name of Child Participant 
 
 
 
Printed Name of Parent/Guardian Participant 
 
 
 
 
 
Signature of Parent/Guardian Participant 
 
 
 
Date 
 
I attest that the individual providing consent had enough time to consider this information, had an 
opportunity to ask questions, and voluntarily agreed to participation in this study and to the child’s 
participation in this study. 
 
 
 
Printed Name of Person Explaining Consent 
 
 
 
 
 
Signature of Person Explaining Consent 
 
 
 
Date 
 
I attest that I or my representative discussed this study with the individual providing consent.   
 
 
 
Signature of Principal Investigator or Sub-Investigator