Omega-3 Supplementation to Both Parent and Adolescent to Reduce Behavior Problems
Phase: PHASE3
Conditions: Behavior Problems
Sponsor: Joint Child Health Project, Mauritius

 
Unique Protocol ID: JCHP-2 
Brief Title: Omega-3 Supplementation to Both Parent and Adolescent   
Official Title: Omega-3 Supplementation to Both Parent and Adolescent to 
Reduce Behavior Problems   
 
Document Date: May 16, 2018 
 
 

 
Experimental Design and Methods 
This section provides the design and method of the overarching study within which the three aims will 
be tested. We also briefly specify further programmatic goals of this line of study, and also how the specific 
expertise of the research team will help ensure feasibility. As with the pilot study, methodology and ultimately 
reporting of results will follow CONSORT 2010 guidelines (Moher et al., 2010). 
Design Overview 
The study design will consist of a stratified, double-blind, placebo-controlled, factorial, randomized 
controlled trial of children and their caregivers in a community sample in Mauritius. This closely follows the 
design of the pilot study (Raine et al. 2015). Unlike the pilot study however, it will utilize a 2x2 factorial design, 
in which children and their parents will be randomly assigned to either omega-3 supplementation (treatment 
condition) or a placebo (control condition), and will have an extended follow-up at 18 months. This results in 4 
groups: (1) child and caregiver both receiving omega-3, (2) child receives omega-3, caregiver receives 
placebo, (3) caregiver receives omega-3, child receives placebo, (4) both parent and child receive placebo. 
Assessments will take place at 0 months (baseline), 6 months (end of supplementation), 12 months (6 months 
post-treatment), and 18 months (12 months post-treatment). 
Background To The Sample and the Mauritius Child Health Project 
Adolescents and caregivers will be drawn from the Mauritius Child Health Study, a multi-generational 
study involving three generations of subjects. Details of these three generations are provided in a cohort profile 
(Raine et al. 2010). Originally set up by Venables (Consultant), this longitudinal study covers early child health 
and development and the identification of early risk factors (including nutrition) for later psychopathology, as 
well as early primary prevention. Early factors that shape later cognitive functioning have also been a focus. 
Given the pilot findings and longitudinal data on the parents, it provides a unique platform on which to build. 
 
Participants, Potential Attrition, and Strategies to Deal with Attrition 
Participants will consist of 400 pairs of children and their caregivers (total N = 800). This replicates cell 
sizes in the pilot data and allows close comparability, with 100 children and 100 adults in each of four cells. 
The caregivers consist of the second generation participants (their parents are the first generation) who 
originally comprised the birth cohort. Their adolescent children are the third generation. All caregivers have 
previously participated in the Mauritius Child Health Project (Raine et al., 2010). Adolescents will be aged 
between 11 and 18 years old, willing to participate in an RCT, and residing in the community. Exclusion criteria 
consist of: (1) allergy to fish or fish products, (2) use of fish oil supplementation in the past six months, (3) 
intellectual disability, (4) participation in the prior RCT. There are currently 1,126 children aged 11-18 years 
actively participating in the Mauritius Child Health Project (51.2% female; 73.2% Indian, 24.5% Creole, 2.3% 
other), 124 of whom took part in the prior RCT and will be excluded. Written informed consent will be obtained 
from the parents with assent obtained from adolescents. The study will take place in interview rooms at the 
project headquarters in Quatre Bornes. 
Attrition is an issue in all RCTs. In our pilot data however, attrition was only 8%, even after one year, 
and is likely attributable both to the long-standing intergenerational relationships between our research team in 
Mauritius and the participants. Attrition may nevertheless be slightly more with the longer follow-up. We have 
three strategies to deal with attrition. First, analyses will be intention-to-treat, including all participants 
regardless of drop-outs. Second, we will enhance participation using staggered incentives for completion. 
Third, we will code for missingness and assess whether it moderates treatment outcome. 
Randomization 
Participants will be randomized into four groups using urn randomization, a covariate adaptive 
randomization method (Wei & Lachin, 1988). Adaptive randomization is employed because it protects the 
study from bias while promoting study objectives, it improves power over standard designs, and it balances 
assignment of treatment across covariates in a way superior to traditional stratification (Rosenberger et al., 
2012). We aim to achieve balanced groups on three main child covariates: age, gender, and ethnicity. An 
advantage of the Mauritius cohort is that we have data on the parents’ externalizing behavior over the life- 
course (Raine et al. 2010) which we could add as a fourth covariate to further ensure that groups are balanced. 
Omega-3 Intervention 

Omega-3 supplementation. As in the pilot study, this consists of a 200 ml drink. The base drink in both 
treatment and placebo conditions consists of fruit juice containing vitamin D (0.85 micrograms) and 
antioxidants (ferric reducing ability of plasma value of 0.71 mmol/100g). For the treatment condition only, 1660 
mg of omega-3 (550 mg of DHA, 550 mg of EPA, 560 mg of alpha-linolenic acid) is added to the base drink. 
Placebo drinks are matched exactly with the fish-oil drink in terms of size, appearance, and flavor. Caregiver 
and child drinks are differentially color-coded. 
This drink is chosen because: (i) it contains a higher dosage of omega-3 than standard capsules in a 
small liquid quantity (60.6% of a standard can of cola) suitable for child / adolescent consumption, (ii) the fruit- 
flavored drink may be better tolerated and result in higher compliance with children than fish-oil capsules. 
Treatment duration and administration. As in the pilot study, treatment duration will be six months. It 
was chosen because prior studies have usually been 2 - 4 months (Sinn et al., 2010), and a longer duration 
may be more effective in producing longer-term behavioral change. The drink will be administered by the 
parents to their children at any time each day. 
Primary Outcome Measures 
The pre-defined primary outcome measures are externalizing behavior problems. Secondary outcomes 
included internalizing behavior problems and neurocognitive / academic mediators. We include additional 
secondary measures of reactive-proactive aggressive behavior and callous-unemotional traits to replicate the 
pilot findings and explore antisocial subtypes (the pilot data showed stronger effects for reactive aggression). 
Externalizing and internalizing behavior problems in children and caregivers will be assessed using the 
Multicultural Family Assessment Module (MFAM - Achenbach et al., 2015) which constitutes counterpart 
scales of the Child Behavior Checklist (CBCL/6-18), Youth Self-Report (YSR), Adult Self-Report (ASR), and 
Adult Behavior Checklist (ABCL). Reliability, validity, and cross-cultural generalizability have been documented 
in over 100 societies (Achenbach & Rescorla, 2001; Achenbach & Rescorla, 2001; Rescorla et al., 2012). The 
Externalizing scale includes Aggressive Behavior and Rule-Breaking Behavior, while the Internalizing scale 
includes Anxious/Depressed, Withdrawn, and Somatic Complaints. Additional syndromes include Attention 
Problems, Social Problems, and Thought Problems. Instruments are also scored on DSM-oriented scales 
comprising items judged by international clinical experts to be very consistent with DSM diagnostic criteria. 
Adolescents will rate themselves on the Youth Self-Report YSR. The caregiver will rate their child on 
the CBCL/6-18, themselves on the ASR, and their spouse on the ABCL. We have established measurement 
invariance of the CBCL in this cohort in Mauritius (Yarnell et al., 2013). Teacher ratings of the child will be 
assessed using the Teacher Report Form of the MFAM. In consultation with Dr. Achenbach (Consultant), we 
will adopt the same system he has used in his national surveys to successfully obtain individual-based teacher 
ratings using payment to the teacher with a parental permission letter (Achenbach & Rescorla, 2001). 
Other measures of antisocial behavior taken in the pilot data will be repeated here to assess 
replicability of original findings. These include the Reactive and Proactive Aggression Questionnaire (RPQ), 
Developed by the PI, this instrument assesses two overlapping yet distinct forms of aggression (Raine et al., 
2006). Psychopathic-like traits will be assessed by caregivers using the Antisocial Personality Screening 
Device (APSD) consisting of three sub-scales: callous-unemotional traits, narcissism, and impulsivity (Frick et 
al., 2000). Due to the introduction of callous-unemotional traits in DSM as a specifier for conduct disorder, 
adolescents will also complete the Inventory of Callous-Unemotional Traits (ICU - Essau et al., 2006). In 
caregivers, psychopathy will be assessed using the self-report Psychopathic Personality Inventory (PPI - 
Lilienfeld & Andrews, 1996). These measures also showed significant changes in the pilot study. 
 
Secondary Outcome Measures 
Neurocognitive Risk Factors. These encompass cognition, emotion, and motor functioning and will be 
assessed using the CANTAB (Robbins et al., 1994). This battery is selected because it is language and culture 
independent, has been extensively validated on children and adolescents, and its user-friendliness to 
adolescents helps assure feasible implementation. Measures are selected based on the most pertinent 
neurocognitive domains relevant to antisocial / aggressive behavior (e.g. Ogilvie et al. 2011; Raine et al., 
2005; Cauffman et al., 2005), including attention (Attention Switching Task), cold executive functioning 
(Stockings of Cambridge), hot executive functioning (Cambridge Gambling Task), emotional decision-making 
(Affective Go/NoGo), social / emotional cognition (Emotion Recognition Task), and impulse control (Stop 
Signal Task). Minimal reading (2 tasks) or no reading (4 tasks) is needed. 

Academic Skills. As per C2’s recommendation for “expanded assessment” to cover other domains, 
academic skills will be assessed using the Wide Range of Achievement Test (WRAT-4 – Wilkinson & 
Robertson, 2006) which provides measures of mathematics, spelling, sentence completion, and word reading. 
Mauritian children today are fluent in English which is the oral and written language of instruction from age 5. 
The parent (CBCL) and youth (YSR) reports also include School Competence scales, while the 
teacher (TRF) form includes Academic Performance and Adaptive Functioning (Achenbach et al., 2015). 
These will extend the scope on mechanistic processes beyond neurocognitive functioning. 
As in the pilot study, parental belief in treatment allocation to the omega-3 group (yes, no) will be 
measured at the end of treatment to assess influence on behavioral change. Demographics will again be 
collected on entry into the study, together with an adverse events questionnaire at the end of treatment. 
Adherence to protocol and blood assays. 
Adherence to the treatment regimen is a frequent problem in RCTs. As before, this will be assessed by 
assays of omega-3 from finger-prick blood taken from both caregiver and adolescent at all four time-points, 
with microwave irradiation assays conducted by team member Dr. Joe Hibbeln an NIAAA. For full 
methodological details see Lin, Loewke, Hyun, Leazer, & Hibbeln (2012). In response to C2, blood will 
additionally be assessed mid-way through supplementation (3 months). To help further enhance adherence, 
participants will be called every two weeks to remind them to use supplements, and to assess frequency of 
drink consumption by caregiver and adolescent in that time period (number of drinks / two weeks). To further 
facilitate compliance, participants will be given a bonus payment if their % blood omega-3 levels have risen 
higher than the bottom quartile of the population. 
Statistical Methods 
Overview. An intention-to-treat (ITT) design using all randomly assigned participants will be employed 
for all data analyses. All data will be blindly double-entered and checked for consistency. 
Aims 1 and 2 (Primary Outcome). These hypotheses will be addressed using adolescent and caregiver 
externalizing responses gathered at baseline and at 6, 12, and 18 month follow-ups. Responses for the 
adolescent will be analyzed separately from responses for the caregiver. For each separate set of responses, 
linear mixed effects models (Molenberghs & Verbeke, 2005) will be used to describe both the pattern of 
response and the within-subject covariance structure across time points. Primary analyses will not include 
baseline covariates, as the randomization will balance the treatment groups and eliminate selection bias. To 
enhance power by controlling for variability in response (Kahan et al., 2014), a small set of baseline covariates 
(e.g. gender, age, ethnicity) will be included in supplementary analyses. 
The response may be transformed (e.g. log transformation) to reduce levels of skewness in the 
response to acceptable levels, as assessed by examination of residuals. Models will include binary indicators 
for adolescent group (omega-3 vs placebo), caregiver group (omega-3 vs placebo), and time (four level factor 
for times 0, 6, 12, and 18), and group by time interaction terms, as fixed effects. Time is modeled as a discrete 
factor to accommodate the patterns observed in our prior study (see Figure under Preliminary Data) where the 
omega-3 adolescent groups are similar at baseline and 6 months, and separate at 12 months. Covariance 
structure models (e.g. random effects, autocorrelated residuals) will be compared using information criteria in 
models containing all main effects and interactions of interest. Once the covariance structure has been chosen, 
the fixed effects will be assessed for significance using likelihood ratio tests. 
Parameter estimates, standard errors, and 95% confidence intervals will be reported. In the case of a 
multiple degree of freedom test (e.g. group by time interaction, with 3 df), single-df contrasts will be estimated 
and reported, to explain the nature of the interaction. 
For the adolescent, Hypothesis 1a will be addressed using a child group by time interaction term (time 
trends for omega-3 and placebo group need not be parallel), or a child group main effect if the interaction term 
is non-significant (parallel time trends). Similar models will address the corresponding Hypothesis 1b for the 
caregiver responses. For both the adolescent and caregiver responses, Hypothesis 2a will be tested by an 
adolescent group by caregiver group by time interaction, or by an adolescent by caregiver interaction if the four 
adolescent by caregiver group trends are parallel. 
Aim 3 (Secondary Outcome). Working hypothesis 3a (change in neurocognitive and academic factors) 
will be tested using the same mixed effects models as outlined above in aims 1 and 2. Working hypothesis 3b 
(mediation), whether improvement in neurocognitive variables (e.g. impulse control) or academic WRAT scores 
partly account for the treatment effect on child behavior will be assessed using mediation analyses. Based on 
the prior study (Raine et al, 2015), effects of supplementation should emerge by the 12-month time point. 
Analyses will test whether changes in risk factors between baseline and the 6 month point mediate the 12- 

month treatment effect. These analyses will be conducted using the PROCESS SPSS macro (Hayes, 2012), 
which will also be used to assess moderated mediation (e.g. neurocognitive measures mediate outcomes more 
strongly in younger adolescents). To test significance of the indirect effects (i.e. mediation), bias-corrected 
confidence intervals for the indirect effects will be generated using 10,000 bootstrap samples. To assess extent 
of mediation, the reduction in variance explained in treatment outcome by the intervention after controlling for 
improved parental behavior (the mediator) will be calculated in a two-step regression. Equivalent analyses will 
be performed for the other neurocognitive variables. 
Moderator Analyses. We will conduct exploratory analyses on age, gender, ethnicity, initial general 
nutritional status, and initial omega-3 levels. To test for moderators, we will extend the models of the primary 
hypotheses to include main effects for each moderator and their interaction effect with the group factor. 
Analyses will initially consider each moderator separately, and multivariate models will examine the joint effects 
of significant moderators. As a secondary analysis we hypothesize that better treatment outcome may be 
observed in children who start out with lower blood levels of omega-3, and have diets high in omega-6 (which 
compete with omgega-3 for uptake).