Fatty Acid Supplements Alter Biological Signatures and Behavior in Children With Autism Spectrum Disorder (ASD)
Phase: PHASE2
Conditions: Autism Spectrum Disorder
Sponsor: Sarah Keim

 
 
 
 
Fatty acid supplements alter biological signatures in children with 
Autism Spectrum Disorder 
 
 
Principal Investigator: Sarah A. Keim, PhD 
Co-Principal Investigator: Lynette K. Rogers, PhD 
 
 
CONFIDENTIAL 
 
 
 
 
 
Version 9 
 
Protocol Version 1 was submitted to the FDA in November 2016 and at initial grant submission. 
Protocol Version 2 was submitted to NIH Nov 2017. No participants were enrolled under 
Protocol Version 1,2,3,4. R66 Phase 1 run under version 5, Aug 2018-Jan 2020. 
 
Version 6 prepared for R33 Phase 2 
 
Version 6 edited to include only the R33 Phase 2 for NCCIH Review (May 6 2020) 
Version 6 edited to respond to feedback from NCCIH (July 30 2020, August 12 2020) 
 
Version 7 created based on final feedback from NCCIH Sept 11 2020 
Version 8 edited to update compensation, statistical considerations, and inclusion criteria (Apr 15 2021) 
Version 9 created to respond to feedback from NCCIH (Oct 20 2021) 
Version 9 edited to increase sample size based on feedback from NCH IRB (Apr 12 2024) 
 
 
 
 
 
 
 
 
 
April 12, 2024 

Fatty acid supplements alter biological signatures in ASD
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STATEMENT OF COMPLIANCE 
 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following: 
 
 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
 
ICH E6; 62 Federal Register 25691 (1997) 
 
NIH Clinical Terms of Award 
 
All key personnel (all individuals responsible for the design and conduct of this study) have 
completed Human Subjects Protection Training. 

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SIGNATURE PAGE 
 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
 
 
 
  
 
 
 
 
*The protocol should be signed by the local investigator who is responsible for the study 
implementation at his/her specific site; i.e., if Investigational New Drug study, the individual who 
signs the Form FDA 1572. 
Site Investigator:*
 
Signed:   
Date: 
Name 
Title
 

Fatty acid supplements alter biological signatures in ASD
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TABLE OF CONTENTS 
Page 
Statement of Compliance ............................................................................................................ ii 
Signature Page .......................................................................................................................... iii 
Table of Contents ....................................................................................................................... iv 
Protocol Summary ...................................................................................................................... 7 
1 
Key Roles ........................................................................................................................... 8 
2 
Background Information and Scientific Rationale ................................................................ 9 
2.1 
Introduction ............................................................................................................. 9 
2.2 
Biological Pathways and Mechanisms .................................................................. 10 
2.3 
Advantages of Fatty Acid Combination Therapies ................................................. 10 
2.4 
Inflammation in Patients with ASD ........................................................................ 11 
2.5 
Evidence for the Need for Early Intervention and Tailored Dosing ......................... 11 
2.6 
Consideration of Comorbidities Common in ASD .................................................. 11 
3 
Study AIMS ...................................................................................................................... 13 
3.1 
Study Outcome Measures ..................................................................................... 13 
4 
Study Design .................................................................................................................... 14 
4.1 
Study Participants ................................................................................................. 14 
5 
Study Enrollment, TREATMENT ASSIGNMENT, and Withdraw ....................................... 15 
5.1 
Enrollment ............................................................................................................ 15 
5.1.1 Medical Record Data ................................................................................. 15 
5.1.2 Recruitment Mailing ................................................................................... 15 
5.1.3 Recruitment Contact .................................................................................. 15 
5.1.4 Treatment Assignment .............................................................................. 16 
5.1.5 Randomization and Masking Procedures .................................................. 16 
5.1.6 Emergency Unblinding .............................................................................. 16 
5.1.7 Withdraw ................................................................................................... 17 
5.1.8 Handling of Withdrawals ............................................................................ 17 
5.1.9 Termination of Study ................................................................................. 17 
6 
Study Procedures and Schedule of Events ....................................................................... 18 
6.1 
Visit 1 (Day 0) ....................................................................................................... 18 
6.2 
E-Visit (Day 25  +/- 7 days phone contact) ............................................................ 19 
6.3 
Visit 2 (Day 45  +/- 14 days) .................................................................................. 19 
6.4 
Visit 3 (Day 90 +/- 14 days) ................................................................................... 20 
6.5 
Additional Routine Communication (throughout participation) ............................... 20 
6.6 
Rationale for Data Collection ................................................................................ 21 
7 
Laboratory evaluations ..................................................................................................... 22 
7.1 
Specimen Preparation, Handling, and Shipping .................................................... 22 
7.2 
Biohazards ............................................................................................................ 22 
8 
Investigational Product ..................................................................................................... 23 
8.1 
Acquisition ............................................................................................................ 23 
8.2 
Formulation, Packaging, and Labeling .................................................................. 23 
8.3 
Dispension ............................................................................................................ 24 
8.4 
Product Storage and Stability ................................................................................ 24 
8.5 
Dosage, Preparation and Administration ............................................................... 24 

Fatty acid supplements alter biological signatures in ASD
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8.6 
Modification of Investigational Product .................................................................. 24 
8.7 
Accountability Procedures for the Investigational Product ..................................... 24 
8.8 
Assessment of Subject Compliance with the Investigational Product .................... 24 
8.9 
Concomitant Medications/Treatments ................................................................... 24 
9 
Risks and benefits ............................................................................................................ 25 
9.1 
Risks ..................................................................................................................... 25 
9.2 
Benefits ................................................................................................................. 26 
10 
Assessment of Safety ....................................................................................................... 27 
10.1 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters ........................................................................................................... 27 
10.2 
Adverse Events ..................................................................................................... 27 
10.3 
Unanticipated Problems ........................................................................................ 28 
10.3.1 Reporting Unanticipated Problems ............................................................ 28 
Incidents or events that meet the OHRP criteria for unanticipated problems will 
be reported using an unanticipated problem report form. The 
unanticipated problem report form will include the following information 
when reporting an adverse event, or any other incident, experience, or 
outcome as an unanticipated problem to the IRB ...................................... 28 
10.4 
Serious Adverse Events ........................................................................................ 29 
10.4.1 Reporting SAE’s ........................................................................................ 30 
10.5 
Procedures in the Event of Abnormal Laboratory Test Values/Clinical Findings .... 30 
10.6 
Type and Duration of Follow-up of Subjects after Adverse Events ........................ 30 
10.7 
Halting Rules ........................................................................................................ 31 
10.8 
Safety Oversight ................................................................................................... 31 
11 
STATISTICAL CONSIDERATIONS .................................................................................. 32 
11.1 
Study hypotheses ................................................................................................. 32 
11.2 
Sample Size Considerations ................................................................................. 32 
11.3 
Planned Interim Analyses (if applicable) ............................................................... 32 
11.3.1 Safety Review ........................................................................................... 32 
11.3.2 Immunogenicity or Efficacy Review ........................................................... 32 
11.4 
Final Analysis Plan ............................................................................................... 32 
12 
Source Documents and Access to Source Data/Documents ............................................ 35 
13 
Quality Control and Quality Assurance ............................................................................. 36 
14 
Ethics/Protection of Human Subjects ................................................................................ 37 
14.1 
Ethical Standard ................................................................................................... 37 
14.2 
Institutional Review Board (IRB) ........................................................................... 37 
14.3 
Informed Consent ................................................................................................. 37 
14.3.1 Informed Consent/Assent Process (in Case of a Minor) ............................ 37 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) .................... 37 
14.5 
Subject Confidentiality .......................................................................................... 37 
14.6 
Study Discontinuation ........................................................................................... 38 
14.7 
Future Use of Stored Specimens .......................................................................... 38 
15 
Data Handling and Record Keeping ................................................................................. 39 
15.1 
Data Management Responsibilities ....................................................................... 39 

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15.2 
Data Capture Methods .......................................................................................... 39 
15.3 
Types of Data ....................................................................................................... 39 
15.4 
Study Records Retention ...................................................................................... 39 
15.5 
Protocol Deviations ............................................................................................... 39 
16 
Publication Policy ............................................................................................................. 41 
17 
Literature References ....................................................................................................... 42 

Fatty acid supplements alter biological signatures in ASD
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PROTOCOL SUMMARY 
 
Title:
Fatty acid supplements alter biological signatures in children with Autism Spectrum 
Disorder 
Background:
Fatty acid supplements are among the most popular complementary products consumed by 
children with Autism Spectrum Disorder (ASD). Their use is driven by anecdotal evidence of 
benefit and a few small RCTs that demonstrated improvements in some externalizing 
behaviors in this population.1-3 Overall, there remains scant evidence for efficacy in treating 
the core symptoms of ASD like social-communication deficits and restricted and repetitive 
behaviors. Furthermore, very little is known about the biological signatures that are affected. 
Over-the-counter (OTC) fatty acid supplements are plentiful and consist of varying 
percentages of gamma-linoleic acid (GLA, an omega-6 fatty acid), eicosapentaenoic acid 
(EPA, an omega-3), and docosahexaenoic acid (DHA, omega-3) and often include vitamins 
or plant extracts as well. Existing RCTs have tested varying combinations of fatty acids with 
conflicting results that likely stem from this variation. Because there are no approved 
medications to treat the core symptoms of ASD, well-intentioned parents are reaching for any 
product that promises improvement in their child, but they are giving these supplements 
without guidance as to their function, mechanism of action, or whether they are harmful or 
helpful in symptom management. 
 
For the proposed project, we will focus on preschool and early school aged children because 
of the extensive literature indicating that a wide range of ASD therapies are maximally 
beneficial if they begin as early as in life as possible after diagnosis. This may be because 
neuroplasticity decreases significantly as children age, accompanied by the fact that DHA 
accretion in the developing brain displays a corresponding decline in rate with age.4 Along 
similar lines, it has recently been reported that fatty acid bioavailability and treatment effects 
in children may depend partially on body size (smaller children exhibit higher fatty acid levels 
for a given dose than larger children). Many pediatric medications are dosed based on body 
weight; however, this concept has been virtually never applied to fatty acid supplementation. 
These gaps in the literature represent a barrier to informing patients and clinicians about 
optimal dosing for a given child, with the end result that some children likely take a dose too 
small to benefit. 
Population: 
A randomized, fully-blind, placebo-controlled trial of 98 children ages 2-6 years with recently- 
diagnosed ASD. The trial will involve 90 days of treatment with a volume of Omega 3-6 product 
providing  50 mg/kg twice per day of GLA+EPA+DHA or (equal volume) placebo and will 
include 4 study visits. 
Study 
Objectives: 
Aim 1. Replicate clinically meaningful changes in the biological signatures (IL-1β, IL-2, TNFα), 
from baseline (day 0) to the end of the 90-day trial. 
Aim 2. Demonstrate that the changes in the biological signatures are associated with 
clinically meaningful changes in ASD symptoms in children per parent report and clinician-
researcher assessment from baseline (day 0) to the end of the 90-day trial. 
 

Fatty acid supplements alter biological signatures in ASD
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1 KEY ROLES 
 
Key Personnel:
Principal Investigator:  Sarah A. Keim, PhD, MA, MS
 
 
Co-Principal Investigator: Lynette K. Rogers, PhD 
 
 
Study Doctor / Co-Investigator:  Daniel L. Coury, MD 
 
Institution: 
 
The Research Institute at Nationwide Children’s Hospital 
Center for Biobehavioral Health 
700 Children’s Drive 
Columbus, OH 43205 
Phone: 614-722-3182 
Fax: 614-722-3544

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2 BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
 
2.1 Introduction 
Although at least 1 in 68 U.S. children has been diagnosed with Autism Spectrum Disorder (ASD), 
no approved medications exist to treat the core symptoms. Atypical antipsychotics are commonly 
prescribed for irritability with ASD, but the side effects are significant, common, and of particular 
concern for very young children.5 Behavioral interventions can offer families approaches to live 
with the atypical behaviors and social deficits of ASD, but they are not cures. Because of the 
dearth of effective treatments, between 52% and 95% of families commonly try complementary 
therapies including fatty acid supplements, based largely on anecdotal evidence. To date, the 
results of efficacy trials testing fatty acid supplementation for ASD are mixed for several reasons 
including variation in the particular combination and dose of fatty acids tested and study design 
shortcomings including lack of blinding or placebo control. Another major reason for the lack of 
clarity is the very limited extent to which studies have evaluated potential biological signatures of 
interest in order to shed light on the pathways through which fatty acids may exert their effect. 
This proposal will determine a primary pathway through which Omega 3-6 supplementation can 
benefit ASD symptoms, by studying a leading candidate set of biological signatures and 
addressing related gaps in the literature that preclude successful efficacy trials. 
 
Fatty acid supplements are among the most popular complementary products consumed by 
children with ASD, driven by anecdotal evidence of benefit and a few small RCTs that 
demonstrated improvements in externalizing behaviors. Because no approved drugs exist to treat 
the core symptoms of ASD, well-intentioned parents are reaching for any product that may help 
their child. The implications are that fatty acid supplements are given to children without any 
evidence base as to their function, mechanism of action, or benefits versus harms. Overall, there 
remains scant evidence for efficacy of fatty acids in treating core ASD symptoms from well- 
designed and adequately-powered RCTs, and very little is known about the mechanism(s) of 
action. Over-the-counter (OTC) fatty acid supplements are plentiful and consist of varying 
percentages of oleic acid (n-9), gamma-linolenic acid (GLA, n-6), eicosapentaenoic acid (EPA), 
and/or docosahexaenoic acid (DHA) and often include vitamins or other plant extracts. Existing 
RCTs have tested varying combinations and doses of fatty acids, and conflicting results likely 
stem from this variation. Recent evidence from our group and others suggest a combination of 
fish and borage oils providing supplementation of DHA, EPA and GLA (“Omega 3-6”) is 
particularly promising compared to other formulations. Based on these foundations, a critical need 
exists to evaluate Omega 3-6 for optimal dosing and changes in a biological signature that are 
related to ASD symptoms, before a rigorous, full-scale efficacy trial can be launched. 
 
Our long-term goal is to identify effective treatments for ASD. The objective of the study is to 
optimize safety and bioavailability of Omega 3-6 supplementation in a unique population of 
children with ASD, evaluate the effect of Omega 3-6 supplementation on biological signatures of 
inflammation, and then relate these changes to changes in core ASD symptoms. Our overall 
hypothesis for the line of research is that Omega 3-6 will alter biological signatures related to 
inflammation in a manner that correlates to ASD-related behaviors. Our hypotheses were 
formulated based on data from our Preemie Tots study, our recently completed Omega Heroes 
trial, and other published data which suggest that the inflammatory markers, IL-1β, IL-2, and 
TNF are consistently elevated in children with ASD and decreases in these markers correlate 
with ASD symptom improvement. The rationale for the proposed research is that its successful 
completion will provide the infrastructure and endpoints necessary to support a future efficacy 
trial to give parents and clinicians unequivocal evidence for fatty acid supplementation for ASD. 

Fatty acid supplements alter biological signatures in ASD
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2.2 Biological Pathways and Mechanisms 
Fatty acids (FA) have diverse and complex biological functions. Long chain polyunsaturated fatty 
acids of the omega-3 series have been extensively studied and demonstrate anti-inflammatory 
properties affecting multiple pathways. The omega-3 DHA comes from fatty fish or algal sources, 
is the primary neuro-active fatty acid in the brain, and is a key player in neurotransmitter function, 
synaptogenesis, gene expression, membrane fluidity, neurogenesis, neuroplasticity, and anti- 
inflammation.6-11 EPA, also from fish oil, is often included in omega-3 fatty acid supplements and 
has triglyceride lowering properties and anti-inflammatory properties similar to DHA. GLA is a 
shorter-chain fatty acid of the omega-6 series derived from plant sources including borage and 
evening primrose. GLA is a precursor to fatty acids such as arachidonic acid (AA) which is 
essential for lipid signaling and hormone synthesis. Biologically available GLA is quickly converted 
to dihomo-γ-linolenic acid (DGLA), which now has been shown to have substantial anti- 
inflammatory properties unique among longer chain n-6 fatty acids.12 As omega-3 and omega-6 
fatty acids share common desaturation and elongation enzymatic pathways, combinations of 
these fatty acids can effectively compete for these enzymes creating a physiologic balance rather 
than a shift toward one pathway or the other. The mechanisms associated with the anti- 
inflammatory effects of long chain fatty acids are still not completely understood but include direct 
binding to g-coupled protein receptor 120 (GCPR120) or peroxisome proliferator-activated 
receptor gamma (PPARγ) to inhibit MAPK or NFkB activation, production of alternative lipid 
products such as resolvins and protectins, and changes in membrane composition that alters 
ligand binding to cytokine or toll-like receptors. Children with ASD have been shown to exhibit 
altered fatty acid metabolism; several investigations have shown decreased levels of DHA, EPA, 
and AA in these children. The reason for these decreases is not completely clear but may be due 
to a combination of diet differences, altered absorption, defects in fatty acid synthesis because of 
polymorphisms in fatty acid desaturase genes (FADS2) or other metabolic alterations.13-15 Since 
all of these FAs contribute to important biological functions, influence anti-inflammatory pathways, 
and enhance anti-inflammatory mechanisms, we speculate that decreases or deficiencies in these 
FAs directly contribute to the increased inflammation observed in ASD patients. 
 
2.3 Advantages of Fatty Acid Combination Therapies 
DHA has been the most widely investigated fatty acid for treatment of mental disorders and has 
demonstrated efficacy in patients with depressive or schizophrenic symptoms. In addition, DHA 
reduces the risk of mild or significant mental delay in extremely preterm infants.16 However, DHA 
alone appears to exhibit little or no benefit in treating aberrant behavioral symptoms.17 Adding 
EPA to DHA (“EPA+DHA,” usually in the form of fish oil) has been evaluated in 4 RCTs for benefits 
to individuals with ASD. One small trial (n=13) showed benefit to language and maladaptive 
behaviors,3 2 other small trials reported benefits limited to hyperactivity,2,18 and the most recent 
trial reported worsening externalizing behaviors.19 EPA+DHA has been shown to be moderately 
efficacious in reducing symptoms of Attention Deficit Hyperactivity Disorder (ADHD) in large RCTs 
(meta-analysis standardized mean difference=0.31, 95% CI: 0.16, 0.47).20 The combination of 
Omega 3-6 holds significant promise to be more efficacious than EPA+DHA for ASD for several 
reasons. Direct comparison of the evidence for Omega 3-6 vs EPA+DHA for treating ADHD in a 
recent meta-analysis reported larger effect sizes and more consistent benefit for omega 3-6 than 
for EPA+DHA (standard difference in means -0.31 favoring omega 3-6-9 (CI: -0.46, -0.16)).21 
Among the largest of these studies, Richardson et al. found a >0.5-standard deviation reduction 
in global Conners’ Teacher Rating Scales scores among children randomized to Omega 3-6 for 
3 months compared to placebo, and Sinn et al. found medium-strong effects of supplementation 
on parent ratings of ADHD symptoms.22,23 However, only 2 placebo-controlled RCTs have tested 
Omega 3-6 formulations for developmental or behavioral outcomes other than ADHD: one study 
demonstrated benefits for  school-age children with Developmental Coordination Disorder on 

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reading and spelling achievement and across multiple behavioral domains, and our Preemie Tots 
study demonstrated reduced autistic behaviors, compared to a placebo.22 
 
2.4 Inflammation in Patients with ASD 
ASD is characterized by pervasive deficits in social communication accompanied by stereotypic 
behaviors. The severity of these symptoms varies widely in ASD and can be accompanied by 
significant cognitive impairment. The etiology of ASD remains unclear, but it has both genetic and 
environmental contributors which impact inflammatory pathways. Inflammation has been well 
characterized in patients with ASD with increases in inflammatory cytokines and chemokines both 
in the circulation and CSF. Interleukin (IL)-1β is an acute response cytokine produced by immune 
cells and acts through targeting cognate receptors present on the cell membrane in tissues. 24 IL-
1β can cross the blood-brain–barrier, inducing increased permeability or be produced by 
astrocytes and microglia in the brain. Increases in IL-1β in neurological tissues, causes increased 
exocytosis of several neurotransmitters, resulting in increased excitability, anxiety, and activation 
of the HPA.25,26 Interleukin (IL)-2 has been extensively studied in the context of brain 
development and T-cell fate.27 IL-2 acts in a trophic manner during development and loss of IL-2 
causes deficits in learning and memory processing in animal studies. Alternatively, IL-2 regulates 
immunological homeostasis, self-tolerance and T cell development and function implying 
dysregulation may play a significant role in neurological auto-immune diseases.24,27  Tumor 
Necrosis Factor alpha (TNF), along with IL-1β, is an acute phase cytokine and is produced in 
response to inflammation.  A recent meta-analysis revealed that TNF was elevated in most 
studies of ASD patients versus typically developing individuals (IL-2 was not evaluated)(Masi et 
al., 2015), and we observed lower levels of IL1β, TNF, IL-2, and IFNγ, in the Omega 3-6 group 
compared to the placebo group at the end of our Preemie Tots trial.  In the R61 phase of the 
current studies we observed that changes in IL1β and TNF approached significance, while IFNγ 
did not show an effect.  Consequently, for the R33 phase we propose to evaluate IL-2, IL-1β, and 
replace INFγ with TNFα as our biological signatures of interest. We will continue to measure INF 
but it will not be a primary focus of the R33. 
 
 
2.5 Evidence for the Need for Early Intervention and Tailored Dosing 
For the proposed project, we will focus on young children ages 2 to 6 because of the extensive 
literature indicating that a wide range of ASD therapies are maximally beneficial if they begin as 
early in life as possible after diagnosis. This may be because neuroplasticity decreases 
significantly as children age, accompanied by the fact that DHA accretion in the developing brain 
displays a corresponding decline in rate with age.4 Along similar lines, it has recently been 
reported that fatty acid bioavailability and treatment effects in children may depend partially on 
body size (smaller children exhibit higher fatty acid levels for a given dose than larger children). 
Many pediatric medications are dosed based on body weight; however, this concept has been 
virtually never applied to fatty acid supplementation. These gaps in the literature represent a 
barrier to informing patients and clinicians about optimal dosing for a given child, with the end 
result that some children likely take a dose too small to benefit. 
 
2.6 Consideration of Comorbidities Common in ASD 
In addition to the social-communication and behavioral features of ASD, many individuals with 
ASD have other comorbidities that impact overall health and daily functioning including at least 
34% have gastrointestinal problems like constipation, 34% are overweight and 18% are obese. 
Theoretically, these conditions may modify the effect of fatty acid supplementation on core ASD 

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symptoms by inhibiting gastrointestinal absorption of the supplement or by sequestering a portion 
of ingested fatty acids in adipose tissue rather having them biologically available. A full-scale 
efficacy trial will likely need to consider these possible treatment modifiers when establishing 
inclusion/exclusion criteria, setting a target sample size, and selecting dose(s). 

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3 STUDY AIMS 
The aims of this project are to: 
 
Aim 1. Replicate clinically meaningful changes in the biological signatures (IL-1β, IL-2, TNF), 
from baseline (day 0) to the end of the 90-day trial. 
Aim 2. Demonstrate that the changes in the biological signatures are associated with clinically 
meaningful changes in ASD symptoms in children per parent report and clinician-researcher 
assessment from baseline (day 0) to the end of the 90-day trial. 
 
 
 
3.1 Study Outcome Measures 
 
Primary Endpoint (Aim 1): The change in each biological signature (IL-1β, IL-2, TNF) from 
baseline (day 0) to the end of the 90-day trial. 
Secondary Endpoint (Aim 2): The change in ASD symptoms per parent report and clinician-
researcher assessment from baseline (day 0) to the end of the 90-day trial. Among the ASD 
outcome measures, the PDDBI autism composite will be of primary interest.  
Exploratory Endpoints: Other ASD outcome measures (Vineland, PLS-5, other PDDBI subscales, 
CARS2, AIM) will be exploratory. 

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4 STUDY DESIGN 
 
4.1 Study Participants 
This single-site study will be conducted at Nationwide Children’s Hospital (NCH). We will recruit 
from among patients who have received an ASD diagnosis by the multi-disciplinary diagnostic 
clinic at the NCH Child Development Center (CDC) within the prior 6 months. We expect large 
numbers of available and interested participants because NCH diagnoses over 800 children with 
ASD each year, and recruitment into past treatment studies in this patient population has been very 
successful. Children evaluated at NCH for an ASD diagnosis undergo a comprehensive 
evaluation with a developmental pediatrician, psychologist, and speech pathologist covering all 
DSM-5 defined ASD criteria, cognitive ability, adaptive behavior, and other behaviors commonly 
comorbid with ASD like hyperactivity, based on trained clinician evaluation and parent report. 
Children who are given the ASD diagnosis, and who meet the other eligibility criteria for the study 
will be recruited. We focus on children in this range to reduce the heterogeneity of the sample and 
to maximize the probability of benefit. 
 
After diagnosis, children will be evaluated for trial eligibility based on medical record review and 
parent interview. Parents will be contacted via letter, phone, and/or email to discuss participation. 
 
Inclusion criteria: age 24 months, 0 days to 83 months, 29 days (2-6 years), ASD 
diagnosis at NCH CDC within prior 6 months, English is primary language. 
 
Exclusion criteria: supplements containing substantial amounts of DHA, EPA or GLA in 
the past 6 months, consume fatty fish >3 times/week, still breast or formula feeding; 
quadriparesis; deafness; seizure disorder, blindness; bleeding disorder; autoimmune 
disorders including Type I diabetes; Fragile X, Rett, Angelman Syndromes; Tuberous 
Sclerosis; feeding problems precluding consumption of the supplement; ingredient allergy; 
planned surgeries scheduled within the timeframe of trial participation. 
 
Additionally, and as part of the informed consent process, Study Staff will ensure that parents 
have a good understanding of study procedures and are able to comply with study procedures for 
the entire length of the study. 

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5 STUDY ENROLLMENT, TREATMENT ASSIGNMENT, AND WITHDRAW 
 
5.1 Enrollment 
The target sample size for this study is 98 children. The study team will be alerted by the electronic 
medical record system when an autism diagnosis is made at NCH for a potentially eligible child. 
The system is updated nightly and study staff will be electronically notified of new diagnoses 
biweekly. Before contacting potential participants for recruitment purposes, the medical record 
will be used to prescreen potentially eligible participants. During this process, study staff will 
examine the ASD diagnostic evaluation to determine if the child meets the inclusion criteria and 
is not excluded due to any exclusion criteria. The medical record will be screened for eligibility 
information, but eligibility will also be confirmed with a legal guardian prior to enrollment. 
 
5.1.1 
Medical Record Data 
The following data items will be collected from the child’s medical record: 
 
Results of the comprehensive ASD evaluation conducted at the NCH Child Development 
Center including scores from developmental and behavioral testing and clinical summary 
notes 
 
Related diagnoses including gastrointestinal disorders and other developmental and 
behavioral diagnoses (e.g., ADHD) and dates of diagnoses 
 
Demographic and participant characteristics (Date of birth, Sex, Race, Ethnicity, Medical 
insurance payor, Language, Life status, Multiple birth, Weight and height from most recent 
clinical encounter and date) 
 
Percent no shows to appointments 
 
Food allergies 
 
Current medication list 
 
Contact information 
 
5.1.2 
Recruitment Mailing 
An introductory letter and informational study brochure will be sent to all eligible families after the 
electronic medical record screening phase. This letter will briefly introduce the study and be 
signed by Dr. Keim and Dr. Rogers. The introductory letter and study brochure will briefly describe 
the purpose of the study and will provide contact information for study staff. This informational 
mailing will request change of address information from the post office to identify families who 
have recently moved. Although we expect up-to-date contact information to be available in the 
electronic medical record due to the recent NCH diagnostic appointment, contact information may 
be verified and/or attempts to locate unreachable families (e.g., those with outdated contact 
information on file) will be made using a web-based online search service that provides a fast and 
effective way to look-up or verify contact information about a person, phone number or address 
of interest. Contact information will be verified using names, addresses, and phone numbers. 
 
 
5.1.3 
Recruitment Contact 
Study staff will phone the family approximately one week after mailing the introductory letter and 
study brochure to further assess eligibility and gauge general interest in participating. Study staff 
will use the phone script devised for this study as a conversation guide. During this phone call, 
study staff will provide a description of the study and its requirements. If the family is interested in 
participating, study staff will describe the study to the legal guardian and ask if the guardian and 
child are interested in coming into Nationwide Children’s Hospital to enroll in the project. During 
this appointment, study staff will meet with the guardian to provide detailed information about the 

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study and answer any questions the guardian may have. If the parent is interested in enrolling 
his/her child and the child meets all eligibility criteria and is not excluded based on the exclusion 
criteria, study staff will guide the parent through the informed consent process. Participation or 
refusal to participate in the study will not affect the child’s entitlement to clinical care. 
This study intends to enroll a special population of children and will accordingly adhere to additional 
protections specified under 45 CFR Part 46 Subpart D – Additional Protections for Children 
Involved as Subjects in Research (45 CFR Part 46.401-409). 
 
5.1.4 
Treatment Assignment 
Study staff will develop a detailed Standard Operating Procedure (SOP) for Randomization and 
Blinding, and Emergency Unblinding Procedures. This SOP will describe how the randomization 
scheme will be generated and that the scheme will be stored in a locked cabinet. 
 
5.1.5 
Randomization and Masking Procedures 
Randomization will involve a stratified block design of randomly varying block sizes of 4 and 6 
with equal allocation to each of the two study arms. A pseudorandom number generator in the 
statistical software package R will be used by Dr. Rausch to implement randomization, and he 
will maintain the randomization sequence such that it is unavailable to other team members. 
 
Randomized block sizes will be generated within each of 4 strata formed by crossing 2 
stratification variables: age (2-3 vs 4-6 years) and sex. This stratification design minimizes 
confounding across groups due to the stratification variables. This block design assures a 
balanced allocation across the 2 study arms. Randomly varying block sizes reduces the chance 
that study staff can guess the next group assignment and thus minimizes unconscious bias in 
patient allocation in the RCT. Furthermore, having an experienced statistician not otherwise 
involved in study operations produce the randomization scheme will help maintain blinding 
which is critical to the integrity of the trial and the value of the findings.  
 
Investigators, staff, parents, and participants will be blind to treatment assignment until all data 
are collected and the database is locked, whereupon only those involved in the analysis will be 
unblinded as needed. Only the statistician (Dr Rausch) will be unblinded during the course of 
the study. 
 
Dr. Rausch will create the randomization scheme in R and upload into the randomization module 
in Redcap. Study staff will use Redcap to select the next sequential ID number and reveal the 
blinded group assignment. The participant will be registered in the electronic medical record 
system and the study doctor will print and sign the prescription. The participant study ID will be 
added to this prescription. Study staff will alert the NCH Investigational Drug Service as to the 
enrollment and randomization of the participant and the participant’s weight. IDS will be given the 
signature page of the consent form and the prescription. IDS will have access to the 
randomization scheme so they can match the animal designation and ID to the appropriate 
treatment and dose. IDS will prepare the prescription and the oil will be distributed directly from 
the pharmacy to the family to prevent unblinding of study staff. 
 
5.1.6 
Emergency Unblinding 
An emergency unblinding SOP also will be generated. Only the IRB or PI are allowed to authorize 
early unmasking in the event of emergency. Otherwise, the randomization assignments will be 
kept secure until closure of the study. 

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5.1.7 
Withdraw 
Study participation will be discontinued under the following circumstances: 
 
1. PI or Study Doctor believe it is in the best interest of the participant; 
 
2. Participant’s legal guardian requests withdraw from the study; 
 
3. Any clinical AE, laboratory abnormality, intercurrent illness, or other medical condition or 
situation occurs such that continued participation in the study would not be in the best 
interest of the child, as determined by the PI and Study Doctor; or 
 
4. Development of any exclusion criteria may be cause for discontinuation. 
 
5. Study instructions are not followed, participation in the study may also be stopped 
 
Withdrawal from the study will not affect the receipt of clinical care at NCH. If the study is halted 
for any reason, participants will be instructed to discontinue taking the assigned treatment and 
return the unused portion to study staff. Participants who choose to discontinue taking the 
assigned treatment during the study will continue to be followed for assessment of the outcomes, 
and their discontinuation will be recorded in the study database. Participants who continue to 
participate but are found to have low adherence to the assigned treatment oil or placebo oil will 
not be withdrawn and will continue to be followed. 
 
5.1.8 
Handling of Withdrawals 
The study will continue to collect safety data on any participant discontinued because of an AE or 
serious adverse event (SAE). If a participant stops taking the assigned oil, the participant will be 
asked to continue scheduled evaluations. 
 
5.1.9 
Termination of Study 
Because interventions based on products with similar composition have not found harm to young 
children, it is not anticipated that the study would need to be terminated due to development of 
toxicities or adverse events due to the intervention. However, the study will be terminated if the 
PI, Study Doctor, IMC, or IRB conclude based on their findings that termination is in the best 
interests of the participants. 

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6 STUDY PROCEDURES AND SCHEDULE OF EVENTS 
Participation will last for 90 days. The study will involve three in-person study visits at 0, 45 
and 90 days and one remote “e-visit” at 25 days. Visit 1 will involve informed consent, 
randomization, gathering anthropometric data, parent completed questionnaires, distribution 
and explanation of the study diaries, a 3 mL blood collection, assessment of ASD symptoms, 
and distribution of the treatment oil/placebo oil. This visit will last approximately 180 minutes. 
Day 25 e-visit will review the study diaries to date, assess compliance, barriers to participation, 
and adverse events. Visit 2 will last about 60 minutes and Visit 3 will last approximately 180 
minutes. They will involve returning all oil bottles distributed at the prior in-person visit, review 
of study diaries, assessment of compliance and adverse events, parent completed 
questionnaires, and a 3 mL blood collection. Visit 3 will assess ASD symptoms as well. In 
addition, study staff will check in with families at four other time points (Day 5 +/-3 days, Day 
35 +/-7 days, Day 60+/-7 days, and Day 80 +/-7 days) to assess compliance, adverse events, 
and barriers to participation. We will ask families for updated contact information at the end of 
each study visit, as well as to provide up to three additional contacts (e.g., friends/family) who 
we may contact if their contact information becomes outdated. 
 
6.1 
Visit 1 (Day 0) 
The enrollment visit will consist of the following activities: 
 
Informed Consent: See Informed Consent section. 
 
Randomization: After consent, families will be randomized. See Randomization and Masking 
Procedures. 
 
Blood Collection: To measure baseline fatty acid levels, a trained pediatric phlebotomist will draw 3 
mL whole blood into a purple top tube. 
 
Anthropometrics:  
Child weight and height will be measured using regularly calibrated SECA scales and stadiometer. 
The weight information will be used to inform IDS in calculating the assigned dose. Both weight 
and height data will be used for exploratory analyses.  On the off chance that a child is not 
cooperative and an accurate weight or height cannot be obtained, staff will use most recent weight 
or height (within previous 4 months) found in the medical record system (EPIC). 
 
Parent Perception of Treatment Group: Parents will be asked what treatment group they believe 
their child will be in to examine the effects of expectation. This information will be gathered using 
a secured survey form for which the response values are inaccessible to study staff until after the 
study is over, to protect blinding. 
 
Barriers to Participation: The family’s intentions to complete the study, attend the next study visit, 
and give their child the assigned oil on a daily basis throughout study duration. 
 
Parent Interview: A parent interview will gather baseline child and family characteristics (sex, age, 
socioeconomic index) to characterize the sample and verify comparability across groups. In 
addition, parents will complete the Baylor fatty acid-specific food frequency questionnaire as an 
interview to measure usual dietary intake of long-chain fatty acids. Finally, parents are asked to 
report on current medications, upcoming surgeries, as well as use of complementary and 
behavioral therapies or programs. 
 
Language skills: Study staff will administer the Preschool Language Scales-5 (PLS-5) with the 
child. 
 

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ASD Assessment: Study staff will observe the child in the lab using the Childhood Autism Rating 
Scale (CARS2) and will interview the parent with the PDD Behavior Inventory (PDDBI). The 
interview will also include Vineland Adaptive Behavior Scales 2nd ed. with a modified timeframe of 
90 days to evaluate adaptive behavior and social communication, the Autism Impact Measure, and 
the “extended” portion of the PDDBI will assess problem behaviors commonly associated with 
ASD which may also be impacted by Omega 3-6. 
 
Gastrointestinal symptoms: Parents will answer a questionnaire about gastrointestinal symptoms 
with the PedsQL Gastrointestinal module (parent report, toddlers/young children versions). 
 
Supplement Dispension: Study staff will counsel families on instructions for taking the daily 
treatment oil/placebo oil, how to record each dose in a provided daily study diary and reporting 
adverse events. IDS will dispense a consistent and ample volume of assigned oil. If child weighs 
more than 32 kg, an extra bottle will be dispensed at Visit 1 and Visit 2. IDS will dispense the 
assigned oil directly to the family and will further instruct on taking the assigned oil. 
 
Compensation: At the conclusion of the visit, participants receive $85 in compensation for their 
time and a Children’s Hospital Parking Garage Pass ($2). Children are given a developmentally- 
appropriate children’s book or small toy (value =$5). 
 
Visit scheduling: Study staff will schedule for in-person visits 2 and 3. 
 
 
6.2 
E-Visit (Day 25 +/- 7 days phone contact) 
This visit will be a remote “E-Visit” via phone and will include the following activities: 
 
Parent Perception of Treatment Group and Adherence: Parents will be asked what treatment 
group they perceive their child to be in to measure potential unblinding of parents, using the 
secured survey form described previously. Additionally, parents are asked to estimate what 
proportion of doses they administered out of the desired number and what proportion of the 
assigned oil they estimate their child usually consumed. Study staff will review study diaries use 
with the parent and will probe for any problems encountered that reflect acceptability, tolerability or 
compliance.  
 
Adverse Events: Parents will be asked if the child has any health or behavior changes since the 
last communication. We will use a modified version of the Safety Monitoring Uniform Report Form 
(SMURF) to document adverse events. 
 
Barriers to Participation: The family’s intentions to complete the study, attend the next study visit, 
and give their child the assigned oil on a daily basis throughout study duration; as well as any 
anticipated barriers to study participation are assessed during each contact with the family. 
 
To simplify our recruitment and retention materials, participants will see that there are 3 in-person 
visits and 5 follow up phone calls. This “E-Visit Day 25” phone call will be one of the five follow- 
up contacts. 
 
6.3 
Visit 2 (Day 45 +/- 14 days) 
This visit will consist of the following activities: 
 
The following activities will follow the same procedures as for Visit 1: Blood Collection, Parent 
Interview, Parent Perception of Treatment Group and Adherence, Adverse Events, Barriers to 
Participation. 
 
Supplement Return: Families will return all previously dispensed oil bottles to IDS for calculating 

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of used/unused portion. 
 
Supplement Dispension: IDS will dispense the assigned oil directly to the family and will further 
instruct on taking the assigned oil. Completed study diary pages will be scanned and diary returned. 
New diary dispensed as necessary. 
 
Compensation: Compensation will be the same as for Visit 1. In addition, families will receive $2 
for every weekly study diary returned. 
 
6.4 
Visit 3 (Day 90 +/- 14 days) 
The final visit will consist of the following activities: 
 
The following activities will follow the same procedures as for Visit 2: Blood Collection, Parent 
Interview, Parent Perception of Treatment Group and Adherence, Adverse Events, Supplement 
Return. 
 
Anthropometrics: Child weight and height will be measured using SECA scales and stadiometer. 
 
Language skills: Study staff will administer the Preschool Language Scales-5 (PLS-5) with the 
child. 
 
ASD Assessment: Study staff will observe the child in the lab using the Childhood Autism Rating 
Scale (CARS2) and will interview the parent with the PDD Behavior Inventory (PDDBI). The 
interview will also include Vineland Adaptive Behavior Scales 2nd ed. with a modified timeframe of 
90 days to evaluate adaptive behavior and social communication, the Autism Impact Measure, and 
the “extended” portion of the PDDBI will assess problem behaviors commonly associated with 
ASD which may also be impacted by Omega 3-6. 
 
Gastrointestinal symptoms: Parents will answer the PedsQL Gastrointestinal Symptoms module. 
 
Supplement Return: Families will return any empty assigned oil bottles and any partially used or 
unopened assigned oil bottles to IDS. 
 
Compensation: Compensation will be the same as for Visit 2, except if families completed all 4 
study visits (1 of which is the E-Visit), they will receive an additional $25 incentive. Families will 
receive $2 for every weekly study diary returned. 
 
6.5 
Additional Compensation 
In addition to the compensation described in sections 6.1, 6.3, and 6.4, we will offer an additional 
incentive for families. The additional incentive will be in the form of a yearly $125 drawing. Enrolled 
participants have the opportunity to receive drawing entries by completing study activities. 
Participants can receive up to 15 entries into the drawing per child. Participants receive one entry 
per child for completing the following activities: 1) return of weekly study diary to study staff; 2) 
return of all used and unused oil bottles at the end of the study.  
 
6.6 
Additional Routine Communication (throughout participation) 
In addition to the study visits, study staff will routinely check in with families at least 5 times (more 
if a family requires additional support) spaced between the visits to assess adherence, adverse 

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events, and barriers to participation. These checks may take place in person, over the phone, or 
through email or text, depending on family preference and ease of communicating. 
 
Families will be reminded of scheduled/upcoming appointments via phone, email and/or text 
(based on family preference). Attempts will be made via phone, email, text, and/or mail to contact 
families who do not show for their scheduled visits. During these communications, both remotely 
and in person, parents may be asked to estimate what proportion of doses they administered out 
of the desired number and what proportion of the assigned oil they estimate their child usually 
consumed. Interim study diary data will be collected via RedCap survey, in the form of photos (of 
each completed study diary page), on paper, or via an app as often as possible from caregivers. 
This will assist in keeping families engaged in study diary use and will provide data if study diaries 
cannot be returned at in- person visits. Study staff will work with the family to increase adherence 
where possible. We will use the modified Safety Monitoring Uniform Report Form (SMURF) to 
document adverse events. Children who do not return for the 2 in-person follow up visits will be 
considered lost to follow-up, but attempts will be made to contact them via phone at these pre-
prescribed intervals. 
 
 
6.7 
Rationale for Data Collection 
Data are collected to: 
 
1) Assess demographic and other participant characteristics, thereby allowing a check of the 
randomization procedure and to report on the composition of the sample; 
 
2) Assess the outcomes of interest; and 
 
3) Determine the feasibility of and aims for the next stage of research. 

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7 LABORATORY EVALUATIONS 
 
7.1 
Specimen Preparation, Handling, and Shipping 
Instructions for the preparation, handling, and storage of specimens are explained clearly in the 
study’s SOP, including required temperatures, aliquots of specimens, where they will be stored, 
and how they will be labeled. Samples will be stored at -80oC until analysis in Dr. Lynette Rogers’ 
laboratory in the Center for Perinatal Research. All laboratory analyses of biospecimens will be 
conducted by Dr Rogers. Serum and RBC samples will be collected from patients at 3 time points 
to determine bioavailability of the supplement in this specific population and to measure the effects 
of treatment on biological signatures. 
 
Fatty acids will be measured in patient red blood cells by standard protocols using gas 
chromatography and will include: capric (10:0), lauric (12:0), myristic (14:0), palmitic (16:0), 
palmitoleic (16:1n-7), steric (18:0), oleic (18:1 n-9), linoleic acid (18:2 n-6), 
-linolenic acid (18:3 n-3) , -linolenic acid (18:3 n-6), arachidonic (AA, 20:4n-6), 
eicosapentaenoic acid (EPA, 20:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3). High and 
low controls will be run with each batch analysis and a run will be considered non-valid if the 
controls are greater than 2 standard deviations from the mean values for DHA, EPA, and GLA. A 
non-valid analysis will be discarded and the samples will be re- analyzed. Biological signatures 
will include the cytokines IL-1β, IL-2,TNFα. These inflammatory cytokines will be measured using 
an ELISA based technology offered by Meso Scale Discovery. Any remaining specimens will be 
maintained in a secured freezer in the Center for Perinatal Research, or Center for Biobehavioral 
Health (depending on space availability) at NCH for future research use. Only the investigators 
for this study will have access to these specimens and permission for future use. The consent 
form for the study will state that leftover specimens may be used for studies of ASD, child 
development or nutrition conducted only by the investigators for the present study. 
 
7.2 
Biohazards 
Blood samples will be obtained from study participants at each visit. This service will be 
provided by the Outpatient Lab at Nationwide Children’s Hospital. As a back-up, Clinical 
Research Services nursing staff can perform this duty. All personnel will be trained in the 
handling of biohazardous samples and are experienced in working with children in these 
situations. Blood samples will be separated prepared for storage by personnel trained in 
handling hazardous biological samples. All procedures will follow Good Clinical Practice and 
federal, state, and institutional regulations and guidelines. 

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8 INVESTIGATIONAL PRODUCT 
 
8.1 
Acquisition 
Nordic Naturals Complete OmegaTM liquid (treatment oil) will be provided by Nordic Naturals 
(Watsonville, CA). Bulk canola oil will be purchased from Welch, Holme and Clark Co., Inc. 
(Newark, NJ). The PI will request an initial supply of the treatment oil and placebo oil to be delivered 
to IDS prior to the study commencing. Additional products will be requested as needed. All 
treatment oil will come from the same manufacturing batch. A sufficient supply of the oils will be 
maintained in IDS throughout the study. 
 
8.2 
Formulation, Packaging, and Labeling 
The treatment oil will be provided by Nordic Naturals in blue glass bottles. It is available over-the- 
counter. The Nordic Naturals Complete OmegaTM liquid contains in each 1 tsp (5ml): 
 
Welch, Holme and Clark Co., Inc. will provide the canola oil placebo. Although fatty acid 
composition varies slightly with each lot of the canola oil, the distributor provides average fatty 
acid composition per 2.5 ml (2.3g) daily serving. Specifically, each 2.5 ml serving of the canola oil 
placebo contains 124 mg of palmitic acid, 39 mg stearic acid, 513 mg linoleic acid, 225 mg 
linolenic acid, and 1346 mg oleic acid. The bulk canola oil placebo will be shipped from the 
distributor in 5-gallon shipments to NCH IDS who will mix the oil with lemon oil at 1:66.6 (1.5 mL 
of lemon oil per 100 mL of canola oil). In this way, the treatment oil and placebo oil will be 
indistinguishable in terms of appearance, smell and taste. IDS will bottle and cap the placebo oil 
in identical bottles and caps as the treatment oil. 

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8.3 
Dispension 
Each participant who is 32 kg or less will receive the same volume of assigned oil at Visits 1 and 
2. An additional bottle will be dispensed at T1 and T2 if participant is over 32 kg. At the initial 
dispension, a prescription will be delivered to IDS, signed by the Study Doctor. At this point, IDS 
will select the appropriate product (Omega 3-6 or placebo), determine the appropriate dose based 
on child’s weight (a volume of Omega 3-6 to provide 50 mg/kg twice per day of GLA+EPA+DHA, 
or equivalent volume of placebo) and place a label conducive to Ohio BOP and FDA regulations 
on each bottle of the treatment oil or placebo oil before being dispensed. Study staff will be unaware 
of the volume prescribed by the pharmacy. The amount of product dispensed to a participant is 
recorded in an overall master log maintained by IDS. No information on the bottles will indicate 
which product is contained in them upon dispension from IDS, and the bottles used for both 
products will be the same and indistinguishable.   
In the case of a participant requiring additional supplies of assigned oil (spillage, losing a bottle), 
interim dispensions can occur via shipping product to the family’s home. One bottle (or more if 
necessary) will be provided in these instances. 
 
8.4 
Product Storage and Stability 
The treatment oil and placebo oil will be kept in a dry, secured environment, controlled and 
monitored for the appropriate temperature. The expiration dates on the products will be monitored 
by IDS to ensure product freshness. The treatment oil and placebo oil will be kept refrigerated to 
maximize freshness. All families will be instructed to keep their bottles refrigerated. 
 
8.5 
Dosage, Preparation and Administration 
Children are randomized to receive a volume of Omega 3-6 (treatment group) that provides 
 50 mg/kg of GLA+EPA+DHA or equal volume of placebo oil based on the child’s weight to be 
administered twice per day by mouth for 90 days. 
 
 
8.6 
Modification of Investigational Product 
No circumstances are known for which the dose should be modified. 
 
8.7 
Accountability Procedures for the Investigational Product 
IDS will monitor the receipt of the treatment oil and placebo oil from the manufacturers, dispension 
of the treatment oil and placebo oil to participants, the return of the treatment oil and placebo oil 
from participants, and the appropriate disposal of any unused assigned oils. 
 
8.8 
Assessment of Subject Compliance with the Investigational Product 
Parents will be provided with a series of diaries and instructions to record whether the child 
consumed the assigned oils each day and what proportion they consumed each day as well as 
any acceptability or tolerability issues. See Study Procedures and Schedule of Events for 
procedures for assessment of compliance. 
 
8.9 
Concomitant Medications/Treatments 
There are no restrictions on what drugs or treatments may be concomitant with the study 
intervention. 

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9 RISKS AND BENEFITS 
 
9.1 
Risks 
All of the research related procedures, surveys, and questionnaires, are designed to meet the 
definition of “minimal risk” in the federal regulations [§45 CFR 46.102(i)] and to be reviewed by 
IRBs under §45 CFR 46.404 “Research not involving greater than minimal risk.” Minimal risk as 
defined in the federal regulations means “that the probability and magnitude of harm or discomfort 
anticipated in the research are not greater, in and of themselves, than those ordinarily 
encountered in daily life or during the performance of routine physical or psychological 
examinations or tests.” In addition, the Study Staff is committed to minimizing risks even when 
the risks are minimal. 
 
The informed consent process will take place in private. Questionnaires will be structured to avoid 
creating discomfort for the research participants; and participants will be reminded at each data 
collection encounter that participation is voluntary, that they have the right to withdraw from the 
research project at any time, and they may refuse to answer or may skip any question. 
 
Experienced pediatric phlebotomists staff will perform biospecimen collections that involve 
minimal discomfort or pain. All staff involved in collection of biospecimens will be credentialed 
accordingly. The physical risks of drawing blood by placing a needle in a vein may cause pain, 
lightheadedness, bleeding, bruising, or swelling at the puncture site. Infection is a rare possibility. 
No alternatives to blood collection exist to reliably assess fatty acid and inflammatory status in 
humans. 
 
The risks of collecting and storing linked clinical data and biospecimens are primarily psycho- 
social. Developing coding strategies to mitigate these risks is a fundamental ethical requirement 
of the study. Potential harm could result from a breach of confidentiality. One of the primary 
concerns is that employment and insurance discrimination might result from exposure of 
information about health history, genetic makeup, or familial predisposition to disease. The risk is 
minor, especially since unique participant identifiers will not be stored with biospecimens. 
 
The Nordic Naturals Complete OmegaTM liquid product contains a combination of fish and borage 
oils. These oils include several LCPUFAs including eicosapentaeonic acid (EPA) and gamma- 
linolenic acid (GLA). Although giving EPA to preterm infants (i.e., children much younger than 
those eligible in this study) has been found to pose some risk related to poorer growth in a single- 
site study that has not been replicated,32 numerous other infancy and post-infancy studies using 
EPA have not reported adverse effects. Because EPA also can increase bleeding time, albeit 
generally clinically insignificantly, children with existing bleeding disorders will be excluded from 
participation. Children with planned surgeries will be excluded. No serious and related adverse 
events related to this treatment were observed in Phase 1 of the trial. 
 
The presence of borage oil in the supplement contributes the GLA content. Most of the safety 
information available about GLA comes from studies that used evening primrose oil, another 
major source of GLA. Animal studies have shown no toxic or carcinogenic effects.33 Previous 
human studies have involved over 4,000 individuals, and no significant adverse effects have been 
reported. There have been two case reports of excessive intake of borage oil or other GLA- 
containing supplements causing worsening seizures in epileptics, but there have been no 
published cases or studies since these reports were published in 1981.34 Although the likelihood 
of this occurring in the present study is very low, children with a seizure disorder will be excluded 
from the study.  A 2004 study of 238 preterm neonates evaluated an infant formula with fish and 

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borage oil compared to a vegetable oil formula fed to infants for 9 months.35 Daily intake amounts 
varied based on volume consumed, but the fish and borage oil group received up to an estimated 
26mg per day of GLA. This is the equivalent of between 3 and 17 mg/kg/day depending on the 
child’s body composition and age. Although Fewtrell and colleagues35 stated adverse events were 
present in both formula groups, the fish and borage oil group experienced more days on ventilator, 
more days on oxygen, and more days with umbilical catheter. Although these adverse events 
were reported, many pre-existed randomization. The infants in that study were already NICU 
patients with multiple co-morbidities present before randomization. Because of the older age of 
the participants in this study, adverse events such as those reported by Fewtrell and colleagues 
are not anticipated.35 
 
The placebo for this study will be canola oil with a natural lemon oil flavoring added. Canola oil is 
a commonly consumed food present in many prepared food products. Whether it is possible to 
have an allergy to canola/rapeseed is controversial, but very rarely some people may have a non-
allergic reaction. Nevertheless, we will exclude any children whose parent states they have 
canola/rapeseed allergy or sensitivity. Lemon oil is Generally Recognized as Safe (GRAS) by the 
FDA. 
 
The study staff will maintain confidentiality of the data in its databases. Data will be summarized 
in aggregate for reporting purposes. Data submitted to the study does include participant 
identifiers, as defined by the U.S. Health Insurance Portability and Accountability Act of 1996 
(HIPAA). The study will follow all necessary measures to assure the participant data stored in the 
database are protected and secure from unauthorized access. 
 
The knowledge to be gained from this investigation outweighs these minimal risks. This study has 
the potential to inform future dietary recommendations for children, especially those diagnosed 
with ASD and associated comorbidities. 
 
9.2 
Benefits 
Omega fatty acid supplementation may benefit participating children by reducing their ASD 
symptomology, and comorbidities. It is not known if these benefits exist. Participation in this study 
will aid in acquiring new knowledge that might help other children with an ASD diagnosis and their 
families in the future. 
 
There are no other known benefits expected from participation in this study. 

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10 ASSESSMENT OF SAFETY 
 
10.1 Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters 
Safety oversight will be under the direction of the Principal Investigators (PIs), Study Doctor, and 
the Independent Study Safety Monitor. All adverse events will be reviewed to rule out the study 
intervention as a potential cause. All Adverse Events will be recorded on a form designated for 
that purpose, labeled with a participant ID and no personal identifiers. For any adverse events 
required to be reported to the IRB or the FDA, and for review purposes by the Independent 
Monitoring Committee (IMC), only the coded ID number will be included in the documentation as 
needed, and no identifiers. The IMC will review adverse events anonymously. If the child’s 
medical record must be accessed to provide additional details as to the event or its treatment, 
study staff will provide abstracted information that is de-identified. Annual reports of findings will 
be submitted to the FDA in line with IND regulations. 
 
 
10.2 Adverse Events 
Adverse Event (AE): ICH E6 defines an AE as any untoward medical occurrence in a patient or 
clinical investigation participant administered a pharmaceutical product regardless of its causal 
relationship to the study treatment. An AE can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally associated with the 
use of investigational product. The occurrence of an AE may come to the attention of study 
personnel during study visits and interviews of a study recipient presenting for medical care, or 
upon review by a study monitor. 
 
All AEs including local and systemic reactions not meeting the criteria for “serious adverse events” 
should be captured on the appropriate CRF. Information to be collected includes event 
description, time of onset, Study Doctor’s assessment of severity, relationship to study product 
(assessed only by those with the training and authority to make a diagnosis, which would include 
MD, PA, Nurse Practitioner, DO, or DDS), and time of resolution/stabilization of the event. All AEs 
occurring during study participation must be documented appropriately regardless of relationship. 
All AEs will be followed to adequate resolution. 
 
Any medical condition that is present at the time that the participant is screened should be 
considered as baseline and not reported as an AE. However, if the medical condition deteriorates 
at any time during the study, it should be recorded as an AE. 
 
All AEs must be graded for severity and relationship to study product. 
 
Severity of Event: All AEs will be assessed by the Study Doctor using a protocol defined grading 
system. For events not included in the protocol defined grading system, the following guidelines 
will be used to quantify intensity. 
 
Mild: events require minimal or no treatment and do not interfere with the participant’s daily 
activities. 
 
Moderate: events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
 
Severe: events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually incapacitating. 

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 
Life threatening: any adverse drug experience that places the participant, in the view of the 
PI, at immediate risk of death from the reaction as it occurred (i.e., it does not include a 
reaction that had it occurred in a more severe form, might have caused death). 
 
Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed. Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 
 
Relationship to Study Products: The Study Doctor’s assessment of an AE's relationship to 
study intervention is part of the documentation process, but it is not a factor in determining what 
is or is not reported in the study. If there is any doubt as to whether a clinical observation is an 
AE, the event should be reported. All AEs must have their relationship to study product assessed 
using the terms: related or not related. In a clinical trial, the study product must always be suspect. 
To help assess, the following guidelines are used. 
 
Related (Possible, Probable, Definite) 
a. The event is known to occur with the study intervention. 
b. There is a temporal relationship between the intervention and event onset. 
c. The event abates when the intervention is discontinued. 
d. The event reappears upon a re-challenge with the intervention. 
 
Not Related (Unlikely, Not Related) 
a. There is no temporal relationship between the intervention and event onset. 
b. An alternate etiology has been established. 
 
10.3 Unanticipated Problems 
The Office for Human Research Protections (OHRP) considers unanticipated problems 
involving risks to subjects or others to include, in general, any incident, experience, or outcome 
that meets all of the following criteria: 
 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the IRB-approved 
research protocol and informed consent document; and (b) the characteristics of the 
subject population being studied; 
 
Related or possibly related to participation in the research (“possibly related” means 
there is a reasonable possibility that the incident, experience, or outcome may have 
been caused by the procedures involved in the research); and 
 
Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
 
 
10.3.1 
Reporting Unanticipated Problems 
Incidents or events that meet the OHRP criteria for unanticipated problems will be reported 
using an unanticipated problem report form. The unanticipated problem report form will include 

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the following information when reporting an adverse event, or any other incident, experience, or 
outcome as an unanticipated problem to the IRB: 
 
Appropriate identifying information for the research protocol, such as the title, 
investigator’s name, and the IRB project number; 
 
A detailed description of the adverse event, incident, experience, or outcome; 
 
An explanation of the basis for determining that the adverse event, incident, experience, 
or outcome represents an unanticipated problem; 
 
A description of any changes to the protocol or other corrective actions that have been 
taken or are proposed in response to the unanticipated problem. 
To satisfy the requirement for prompt reporting, unanticipated problems will be reported using 
the following timeline: 
 
Unanticipated problems that are serious adverse events will be reported to the IRB, 
Independent Safety Monitor(s), and NCCIH within 7 days of the investigator becoming 
aware of the event. 
 
Any other unanticipated problem will be reported to the IRB, Independent Safety 
Monitor(s), and NCCIH within 14 days of the investigator becoming aware of the 
problem. 
All unanticipated problems should be reported to appropriate institutional officials (as required 
by an institution’s written reporting procedures), the supporting agency head (or designee), and 
OHRP within one month of the IRB’s receipt of the report of the problem from the investigator. 
 
10.4 Serious Adverse Events 
Serious Adverse Event (SAE): An SAE is defined as an AE that meets one of the following 
conditions: 
 
 
Death during the period of protocol defined surveillance. 
 
Life-threatening event (defined as a subject at immediate risk of death at the time of the 
event). 
 
An event requiring inpatient hospitalization or prolongation of existing hospitalization 
during the period of protocol defined surveillance. 
 
Results in congenital anomaly or birth defect. 
 
Results in a persistent or significant disability/incapacity. 
 
Any other important medical event that may not result in death, be life threatening, or 
require hospitalization, may be considered a serious adverse experience when, based 
upon appropriate medical judgment, the event may jeopardize the subject and may require 
medical or surgical intervention to prevent one of the outcomes listed above. Examples of 
such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 

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The Study PI and Independent Monitoring Committee will be responsible for determining 
whether an SAE is expected or unexpected. An adverse event will be considered unexpected if 
the nature, severity, or frequency of the event is not consistent with the risk information 
previously described for the intervention. 
 
All SAEs will be: 
 
Recorded on the appropriate SAE CRF. 
 
Followed through resolution by a Study Doctor. 
 
Reviewed and evaluated by a Study Doctor. 
 
10.4.1 
Reporting SAE’s 
Any AE considered serious by the PI or Study Doctor or which meets the aforementioned criteria 
must be submitted on an SAE form to the NCH IRB within 3 business days of discovery. All SAEs 
will be followed until satisfactory resolution or until the Study Doctor deems the event to be chronic 
or the patient to be stable. 
 
Annual reports of findings will be submitted to the FDA in line with IND regulations. 
 
SAEs that are unanticipated, serious, and possibly related to the study intervention will be 
reported to the Independent Monitors(s), IRB, FDA, and NCCIH in accordance with 
requirements.  For the IND: 
 
7-day IND Safety Report (unexpected fatal or life-threatening AEs related to the 
intervention); a copy of the report sent to the FDA will be submitted to the NCCIH 
Program Officer and Independent Safety Monitor(s) within 24 hours of FDA notification. 
 
15-day IND Safety Report (any other serious and unexpected AE related to the 
intervention); a copy of the report submitted to the FDA will be submitted to the NCCIH 
Program Officer and Independent Safety Monitor(s) within 24 hours of FDA notification. 
 
All other AEs documented during the course of the trial will be reported to NCCIH on an 
annual basis by way of inclusion in the annual report and in the annual AE summary 
which will be provided to NCCIH and to the Independent Monitors. The Independent 
Safety Monitor(s) Report will state that all AEs have been reviewed. 
 
10.5 Procedures in the Event of Abnormal Laboratory Test Values/Clinical Findings 
Collection of laboratory data should be limited to those laboratory parameters that are relevant to 
safety, study outcome measures, and/or clinical outcome. No known fatty acid value is considered 
to be abnormal or to present a safety concern. As a result, it is not anticipated that any abnormal 
laboratory values will be found. However, any abnormal clinical findings will be reported to the 
child’s physicians. 
 
10.6 
Type and Duration of Follow-up of Subjects after Adverse Events 
Unanticipated problems will be recorded in the data collection system throughout the study. 

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The PI will record all reportable events with start dates occurring any time after informed consent 
is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study 
participation. At each study visit, the investigator will inquire about the occurrence of AE/SAEs 
since the last visit. Events will be followed for outcome information until resolution or stabilization. 
 
10.7 
Halting Rules 
If it is found that the study intervention is related to any serious adverse events, enrollment would 
be temporarily suspended until a safety review is convened, the objective of which is a decision 
as to whether the study (or intervention for an individual or study cohort) should continue per 
protocol, proceed with caution, be further investigated, be discontinued, or be modified and then 
proceed. Suspension of enrollment (for a particular group or for the entire study) is another 
potential outcome of a safety review. 
 
Subsequent review of serious, unexpected, and related AEs by the PI, Study Doctor, the 
Independent Study Safety Monitor, the IRB, or the FDA or relevant local regulatory authorities 
may also result in suspension of further trial interventions/administration of study product. The 
FDA retains the authority to suspend additional enrollment and study interventions/administration 
of study product for the entire study, as applicable. 
 
Based on the large number of previous studies that have utilized a similar intervention in children 
and the widespread use of the study products in the U.S. consumer marketplace, there are no 
expectations for halting the study enrollment or intervention due to safety concerns. However, it 
will be left to the judgment of the PI with the advice of the Study Doctor and Safety Monitor whether 
a pattern of adverse events or other circumstances warrant halting. 
 
10.8 
Safety Oversight 
Safety oversight will be under the direction of the PIs and Independent Study Safety Monitor. All 
adverse events will be reviewed to rule out the study intervention as a potential cause. 

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11 STATISTICAL CONSIDERATIONS 
 
11.1 
Study hypotheses 
This study will test the hypothesis that Omega 3-6 will produce a clinically significant reduction 
(improvement) in plasma levels of IL-1β, IL-2, or TNFα compared to placebo and such changes 
will be associated with clinically meaningful changes in ASD symptoms. 
 
11.2 
Sample Size Considerations 
Our power analysis is based on a comparison on pre-post change between all placebo group 
participants and the treatment group participants receiving Omega 3-6. We use a Type I error 
rate of .05 to test our primary aim that given inflammatory markers will demonstrate 
improvements from baseline to posttest compared to the placebo group. 
 
For the purposes of examining power, we assume an independent samples t-test on pre-post 
change with ≥80% power to obtain a sufficient per group sample size (n), equal across groups. 
Consequently, the standardized mean difference ) is the effect size of interest. We base our 
estimate of the intervention effect size on our recently completed Omega Heroes trial where the 
detected effect on IL-2 was 0.62. Accordingly, N=98 (N=84 with complete data combined with the 
assumption of 12% loss to follow-up) provides adequate power to detect this effect size. Furthermore, 
this effect size (effect on inflammation) is clinically meaningful, based on the meta-analysis by 
Masi which compared children with ASD to typically-developing children and observed differences 
in cytokine levels of  and 1.0 for 2 of our biomarkers of interest.41 
 
The sample of N=98 (N=84 with complete data combined with the assumption of 12% loss to 
follow-up) will also provide >80% power to detect modest correlations (r=0.30) between changes 
in the biological signatures and ASD symptoms. 
 
11.3 
Planned Interim Analyses (if applicable) 
No interim analysis is planned. 
 
11.3.1 
Safety Review 
Based on the large number of previous studies that have utilized the same intervention in similar 
populations and the widespread use of the study products in the U.S. consumer marketplace, 
there are no expectations for halting the study enrollment or intervention due to safety concerns. 
However, it will be left to the judgment of the Principal Investigator with the advice of the IMC 
whether a pattern of adverse events or other circumstances warrant halting. See the section 
“Assessment of Safety” for details about safety monitoring. 
 
11.3.2 
Immunogenicity or Efficacy Review 
None 
 
 
11.4 
Final Analysis Plan 

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Aim 1 Primary Analysis: To formally examine the impact of the intervention, the primary analysis 
will be to compare the Omega 3-6 treatment group to the placebo group on the change in each 
inflammatory marker from baseline (day 0) to the end of the trial (day 90). 
 
Aim 2 Secondary analysis: Examine correlations between changes in each inflammatory marker 
and changes in ASD symptoms based on the PDDBI autism composite.  
 
Exploratory analyses: Examine sex and age as potential treatment effect modifiers for the analyses 
pertaining to Aims 1 and 2. Examine correlations between changes in each inflammatory marker and 
changes in ASD symptoms based on the other ASD outcome measures (Vineland, PLS-5, other 
PDDBI subscales, CARS2, AIM) for the whole sample and also consider child sex and age as 
potential treatment effect modifiers. Examine differences in the change in Omega 3-6 bioavailability 
and the biological signatures from baseline (day 0) to the end of the 90-day trial by the presence of 
gastrointestinal problems and degree of child adiposity (baseline BMI z-score and BMI z >85th 
percentile) at baseline (day 0). 
 
 
The primary approach for the analyses is intent-to-treat: individuals will be kept in the group to 
which they were randomized, regardless of protocol violations or drop-out. Every effort will be 
made to minimize missing data. Analyses will explore plausible missing data mechanisms, and 
baseline predictors of missingness will be investigated. Also, when possible maximum likelihood 
estimation (MLE) will be employed where predictor variables associated with missingness will be 
investigated in sensitivity analyses. MLE generally provides valid inferences when the missing 
data mechanism is missing at random37 and is more appropriate than many commonly employed 
approaches to data imputation.38 
 
For Aim 1, for the Primary Analysis to formally examine the efficacy of the intervention on 
inflammatory markers, we will compare the Omega 3-6 treatment group   to the placebo group   
on pre-post change in each inflammatory marker. The following is an illustration of our statistical 
model for repeated measures (RM), similar to analysis of covariance (ANCOVA) but based on  
maximum  likelihood  estimation,  that  will  be  employed  for  these      analyses39: 
yit   0  1Timet   2Timet  Trti   it 
, where implicitly it is assumed that there is no main effect of Trti 
, yit is the value for a given inflammatory marker for individual i at time t,  Timet is coded as 0 for 
the pretest and 1 for the 90-day post-treatment, and the primary effect of interest is 2 
, which 
corresponds to the difference between the groups on change in the inflammatory marker of 
interest. In this RM model, the baseline measure of the individual’s inflammatory marker is 
constrained to have equal means across groups by assuming there is no main effect for Trti . This 
constraint yields a statistical test for 2 analogous to the test of the treatment group main effect 
in an ANCOVA covarying the pretest measure of the outcome variable. The main advantage of 
the RM model is that  it  can be estimated in  SAS  PROC MIXED using     maximum  likelihood 

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estimation on all available data by treating Timet as a repeated measures factor with 2   levels, 
whereas an ANCOVA model would require listwise deletion for missing data.38 
 
 
 
For Aim 2, we will calculate and statistically test Pearson correlations between changes in each 
inflammatory marker and changes in ASD symptoms using the PDDBI autism composite, from 
baseline (day 0) to the end of the trial (day 90). If substantial missing data is realized, a mixed-
effects model will be employed to examine each of these correlations.  
 
Exploratory analyses:  
We will conduct exploratory analyses to examine sex and age as potential treatment effect 
modifiers for the analyses described above for Aim 1 by testing an interaction term in the model 
(p<.05 will indicate significant effect modification). 
 
We will repeat the analyses described above for Aim 2, but now for the other ASD outcome 
measures (Vineland, PLS-5, other PDDBI subscales, CARS2, AIM). Child sex and age will be 
examined as potential treatment effect modifiers by testing interaction terms in a regression 
model (p<.05 will indicate significant effect modification). 
 
We will employ linear regression models to examine associations between ASD co-
comorbidities at baseline (day 0) including obesity (e.g., age and sex-specific BMI-z 
(continuous) and BMI z >85th percentile (binary)) and gastrointestinal problems at baseline (day 
0) with the change in Omega 3-6 bioavailability and the change in each inflammatory marker 
from baseline (day 0) to the end of the 90-day trial. 
 

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12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS 
The study will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of subjects. Only authorized Study Staff and NCH IRB staff will have access to the 
documents. 
 
Source data are all information, original records of clinical findings, observations, or other activities 
in a clinical trial necessary for the reconstruction and evaluation of the trial. Examples of these 
original documents and data records include, but are not limited to, caregiver completed 
questionnaires/interviews, hospital records, clinical and office charts, laboratory notes, 
memoranda, subjects’ memory aid 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, and subject files and records kept at the pharmacy, at the laboratories, and medico- 
technical departments involved in the clinical trial. It is acceptable to use CRFs as source 
documents. 

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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
The quality management program for this study will consist of a system of quality checks on all 
data collection procedures in addition to a system of training. 
 
All study staff that will have contact with study participants will be trained in accordance with the 
gold standard for each data collection method. At periodic intervals the quality of the data 
collections being performed will be evaluated by direct observations by the PI or a designated 
subject matter expert. All paper-based data collection will be evaluated for protocol compliance, 
completeness, and accuracy by the PI via direct observation of Study Staff and checks of the 
completed forms. Data entry quality will be assessed by a random check of 10% of the data 
entries. Laboratory quality control and assurance procedures will follow SOP already in place in 
the laboratory and used during previous studies. 

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14 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
 
14.1 
Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the principles set 
forth in The Belmont Report: Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research of the US National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR Part 46 and/or the 
ICH E6; 62 Federal Regulations 25691 (1997). 
 
14.2 
Institutional Review Board (IRB) 
The Nationwide Children’s Hospital IRB will review and approve the protocol for this study 
including the associated informed consent documents and recruitment materials. Any 
amendments to the protocol or consent materials will also be approved before they are placed 
into use. 
 
14.3 
Informed Consent 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continuing throughout the individual’s study participation. Discussion of risks and 
possible benefits will be conducted with the participants and their families. Consent forms 
describing, in detail, the study interventions/products, study procedures, and risks are given to 
the family and written documentation of informed consent is required prior to starting 
intervention/administering study product. Consent forms will be IRB-approved and the parent will 
be asked to read and review the document. Attempts will be made to have a second caregiver (if 
available) complete a consent form as well to work to build buy-in from second caregivers. Upon 
reviewing the document, Study Staff will explain the research study and answer any questions that 
may arise. The parent will sign the informed consent document prior to randomization and any 
procedures being done specifically for the study. The parent will have the opportunity to discuss 
the study with his/her family or think about it prior to agreeing to participate. The participant may 
withdraw consent at any time. A copy of the informed consent document will be given to the family 
for their records. The rights and welfare of the participants will be protected by emphasizing to 
them that the quality of their medical care will not be adversely affected if they decline to participate 
in this study. 
 
14.3.1 
Informed Consent/Assent Process (in Case of a Minor) 
Children may participate in this study under the written informed consent provided by a parent or 
legal guardian. 
 
Children of a parent who is cognitively impaired or mentally ill are not eligible if the parent is not 
able to understand fully the research project and to grant informed consent. 
 
14.4 
Exclusion of Women, Minorities, and Children (Special Populations) 
The study will not exclude any special populations. 
 
14.5 
Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s) and their agents. This confidentiality is extended to cover testing of biological samples 
and genetic tests in addition to the clinical information relating to participating subjects. 

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The study protocol, documentation, data, and all other information generated will be held in strict 
confidence. No information concerning the study or the data will be released to any unauthorized 
third party. 
An Independent Study Safety Monitor or other authorized representatives may inspect all 
documents and records required to be maintained, including but not limited to, data collection 
records and pharmacy records for the subjects in this study. Records will be retained indefinitely. 
 
14.6 
Study Discontinuation 
In the event that the study is discontinued, participants may discontinue use of the treatment oil 
and placebo oil safely.  There is no known risk to discontinuation. 
 
14.7 
Future Use of Stored Specimens 
Any residual specimens will be maintained after the study is complete and will be available for 
future analysis and use, per the participant informed consent document. Specimens may be 
analyzed for future studies of child nutrition, neurodevelopment and behavior and growth. 
Specimens will be maintained in a secure location in the Center for Perinatal Research or Center 
for Biobehavioral Health at NCH and will be labeled with a numeric code and no identifying 
information. This is noted on the consent form and additional consent to bank leftover 
biospecimens in this manner will be obtained from participating families. Only the investigators for 
this study will have access to these specimens and permission for future use. 

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15 
DATA HANDLING AND RECORD KEEPING 
The PI is responsible to ensure the accuracy, completeness, legibility, and timeliness of the data 
reported. All source documents should be completed in a neat, legible manner to ensure accurate 
interpretation of data. Dark ink is required to ensure clarity of reproduced copies. When making 
changes or corrections, cross out the original entry with a single line, and initial and date the 
change. 
 
Copies of the CRF will be used as source documents and maintained for recording data for each 
participant enrolled in the study. Data reported in the CRF derived from source documents should 
be consistent with the source documents or the discrepancies should be explained. 
 
15.1 
Data Management Responsibilities 
All source documents and laboratory reports will be reviewed by the investigator team and data 
entry staff, who will ensure that they are accurate and complete. Adverse events must be graded, 
assessed for severity and causality, and reviewed by the PI or designee. 
 
Data collection is the responsibility of the Study Staff under the supervision of the PI. During the 
study, the investigator must maintain complete and accurate documentation for the study. 
 
The Study Staff is also responsible for data management, quality review, analysis, and reporting 
of the study data under the direction of the investigator team. 
 
15.2 
Data Capture Methods 
For the purposes of this pilot study, data capture will be via paper and electronic methods (e.g., 
Red Cap). The data entry system will include password protection and internal quality checks, 
such as automatic range checks and data validation, to identify data that appear inconsistent, 
incomplete, or inaccurate. Clinical data will be entered directly from the source documents. 
 
15.3 
Types of Data 
Data for this study will include questionnaires, interviews, medical record, and laboratory 
measures. 
 
15.4 
Study Records Retention 
Study records will be kept in a secure, locked location in the Center for Biobehavioral Health at 
NCH and kept per procedures laid out in federal regulations. 
 
15.5 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or Manual of Procedures requirements. The noncompliance may be either on the part of 
the subject, the investigator, or the Study Staff. As a result of deviations, corrective actions are to 
be implemented promptly. 
 
These practices are consistent with ICH E6: 
 
4.5       Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
5.1       Quality Assurance and Quality Control, section 5.1.1 
5.20     Noncompliance, sections 5.20.1, and 5.20.2. 

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All deviations from the protocol must be addressed in study subject source documents and must 
be maintained in the regulatory file, as well as in the subject’s source document. Protocol 
deviations must be sent to the local IRB per their guidelines. 

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16 
PUBLICATION POLICY 
Following completion of the study, the PI is expected to publish the results of this research in 
scientific journals. The International Committee of Medical Journal Editors (ICMJE) member 
journals have adopted a trials-registration policy as a condition for publication. This policy requires 
that all clinical trials be registered in a public trials registry such as ClinicalTrials.gov, which is 
sponsored by the National Library of Medicine. Other biomedical journals are considering 
adopting similar policies.  The PI will register this trial in an acceptable registry. 

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17 
LITERATURE REFERENCES 
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