Sulforaphane in Autism: A Treatment Trial to Confirm Phenotypic Improvement With Sulforaphane Treatment in a New Jersey (NJ) Population of Individuals With Autism
Phase: PHASE2
Conditions: Autism, Autistic Disorder, Autism Spectrum Disorder, Autistic Behavior
Sponsor: Rutgers, The State University of New Jersey

Document type: Protocol and Statistical Analysis Plan 
Short Title: Sulforaphane in a New Jersey (NJ) Population of Individuals With Autism 
Official Tittle: Sulforaphane in Autism: A Treatment Trial to Conﬁrm Phenotypic 
Improvement With Sulforaphane Treatment in a New Jersey (NJ) Population of 
Individuals With Autism 
ClinicalTrials.gov Identiﬁer: NCT02677051 
Document date: 07/25/2023 

Protocol Version 9.0 -04/15/2020
Department of Statistics
Hill Center, Room 501
Rutgers, The State University of New Jersey
110 Frelinghuysen Road
Piscataway, NJ 08854-8019
stat.rutgers.edu
office@stat.rutgers.edu
p. 848-445-2690
f. 732-445-3428
Title of Project:
Sulforaphane in autism. A treatment trial to confirm 
phenotypic improvement with sulforaphane 
treatment in a NJ population of individuals with 
autism.
Principal Investigator:
Steve BuyskePhD.
Funding Source(s):
The Governor's Council for Medical Research and 
Treatment of Autism of New Jersey
1. Purpose/Specific Aims
To complete a trial with sulforaphane in a NJ cohort of individuals with autism with 
the goal of independently replicating the findings that sulforaphane treatment is 
associated with improvement of core symptoms in autism. 
Specific Aims:
Aim 1: To perform a double blind randomized control pilot study with sulforaphane in 
a NJ cohort of autism of at least 40 individuals with autistic disorder completing the 
study to replicate the previous findings of the Singh study. 
Aim 2: To genotype specific functional variants in genes of sulforaphane metabolism 
to use as covariates to account for potential variability in treatment response.
Aim 3: To measure the pre- and post-treatment glutathione levels and glutathione 
status (e.g. how much is oxidized or reduced) to use as a covariate to account for 
potential variability in treatment response.
1.1
Objectives 
The objectives of this study are:

To replicate the study of Singh et al (Singh, Connors et al. 2014), that 
sulforaphane treatment results in improvements in core symptoms in 
autistic disorder (ClinicalTrials.gov Identifier NCT01474993). 

To account for potential variability in treatment response due to genetic 
variations in sulforaphane metabolizing enzymes.

To correlate pre- and post- treatment glutathione levels with potential 
variability in treatment response. 
1.2
Hypotheses 
That treatment with sulforaphane will improve symptoms of autistic disorder as 
measured by the Aberrant Behavioral Checklist (ABC), the Social Responsiveness 
Scale (SRS) and the Clinical Global Impressions test (CGI).  
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2. Background and Significance
One of the most promising findings in autism recently is that of Singh et al. (Singh, 
Connors et al. 2014) which reported results on a double blind study with the 
isothiocyanate, sulforaphane. They reported improvement by multiple metrics. 
Significant improvement was seen in behavior as measured by the Aberrant Behavioral 
Checklist (ABC) and by the Social Responsiveness Scale (SRS).  In addition a 
significantly greater number of participants receiving sulforaphane had improvement in 
social interaction, abnormal behavior, and verbal communication as per the Clinical 
Global Impression (CGI). The Singh study needs to be replicated in an unrelated 
dataset to help determine if their results represent a real phenomenon and if a large 
scale clinical trial is warranted. In addition, there was some variability in response to 
treatment. We will attempt to replicate their study and try to account for some variability 
in response to sulforaphane treatment. The Singh study also demonstrated that 
treatment in autistic disorder using orally administered sulforaphane was well tolerated 
and safe. 
Sulforaphane (1-isothiocyanato-4R- (methylsulfinyl)butane) is an isothiocyanate derived 
from the action of the plant enzyme myrosinase on glucosinolates including 
glucoraphanin.  Cruciferous vegetables are an important source for glucosinolate and it 
is present at high levels in broccoli sprouts (Fahey, Zhang et al. 1997). We propose to 
administer Avmacol®. As far as we know Avmacol® is the only available product on the 
consumer market which co-delivers active myrosinase (a plant enzyme) along with its 
substrate (glucoraphanin), and is shelf-stable at room temperature.  Avmacol® is a 
commercially-available over the counter supplement that is presently produced and 
marketed by Nutramax Laboratories, Inc., Edgewood, MD, USA, and has been on the 
market since 2013. 
Sulforaphane is the most potent naturally occurring inducer of mammalian 
cytoprotective enzymes. Its therapeutic potential is based at least in part on their ability 
to upregulate genes responsible for removal of endogenous and exogenous 
electrophiles and to regulate immune and inflammatory genes through the Keap1–Nrf2 
pathway. The transcription factor Nrf2 (Nuclear factor-erythroid factor 2) is an important 
regulator of a large fraction of cytoprotective gene products, and it orchestrates 
protective responses to a diversity of endogenous and exogenous stresses including 
those involved in neurodegenerative diseases (Gan and Johnson 2014). Importantly, 
Nrf2 levels are substantially depressed in ASD for 45% of typically developed children. 
An important subset of these upregulated genes is that of the glutathione (GSH) 
metabolism genes some of which are relevant in sulforaphane metabolism. 
Sulforaphane is metabolized by glutathione-S transferases (GST's) through conjugation 
to GSH. Plasma levels and excretion of sulforaphane have been reported to be 
significantly different based on the presence or absence of the null genotype of GSTM1 
(Gasper, Al-Janobi et al. 2005, Lampe 2007, Steck, Gammon et al. 2007). In turn, 
GST's as well as other genes of glutathione metabolism and possibly other relevant 
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phase II genes, are upregulated by sulforaphane. The NAT2 slow-metabolizing 
genotype along with GSTM1 null modifies benefit of cruciferous vegetables. Sex 
specific differences in GSTA1 up-regulation during cruciferous vegetable diet with 
GSTM1- and GSTT1- null genotypes has been described. It was postulated that these 
Null genotypes result in slower metabolism and therefore result in a stronger up-
regulation of NRF2. GST's have overlap of substrate and it is hypothesized that the 
GSTT1 enzyme has little effect in GSTM1- not null individuals but is more important in 
individuals who are GSTM1-null.  GSTP1 and GSTA1 may also be important in 
sulforaphane metabolism.  It is therefore possible that genotypes of GST's that 
metabolize sulforaphane may contribute to the variability seen in the phenotypic 
response to sulforaphane treatment. 
Interestingly GST's and other glutathione metabolism genes have been reported as risk 
factors for autism. We previously reported evidence that GSTM1 and glutathione 
peroxidase-1 (GPX1) are risk factors for autism (Buyske, Williams et al. 2006, Ming, 
Johnson et al. 2009). Others reported data that support our GSTM1 association(James, 
Melnyk et al. 2006). Other genes of glutathione metabolism implicated in autism by us 
and others include GSTP1 (Williams, Mars et al. 2007), cystathionine gamma-lyase 
(CTH), the catalytic subunit of glutamate-cysteine ligase, catalytic subunit (GCLC), 
glutaredoxin-3 (GLRX3), alcohol dehydrogenase-5 (ADH5) and glutaredoxin (GLRX). 
Other important oxidative stress related genes that have been associated with autism 
are paraoxonase 1 (PON1) and glyoxalase 1 (GLO1). Interestingly, both enzymes are 
GSH dependent.
Oxidative stress and autism:
Inflammation and systems to combat oxidative stress has been repeatedly implicated in 
autism, including altered enzyme levels, altered metabolite and biomarker levels and 
genetic risk factors. Enzyme activity of several key anti-oxidant genes have been 
reported altered in autism including reduced enzymatic activities of glutathione 
peroxidase (GPX), superoxide dismutase (SOD) and catalase. Another study reported 
significantly reduced levels of SOD and GPX and increased levels of malondialdehyde 
in subjects below the age of 6 but not above. Reduced taurine levels as well as 
carnosine levels have been reported (Ming, Stein et al. 2012). In addition, elevated 
nitrite concentrations have been detected in individuals with autism along with t-bars 
and xanthine oxidase activity in red cells.  Increased t-bars, Na+/K+-ATPase, 
isoprostanes and hexanoyl-lysine adduct (HEL) have also been reported.  Increased 
non-protein-bound iron and 4-hydroxynonenal protein adducts (4-HNE PAs) were also 
reported increased in autism. 
Glutathione (GSH) levels have been shown to be altered in autism. Total GSH was 
reportedly decreased while plasma levels of GSSG were elevated; the tGSH:GSSG 
ratio was low (James, Cutler et al. 2004). A meta-analysis of oxidative stress-related 
biomarkers and genes showed associations with autism for levels of methionine, 
cysteine, and GSH, glutathione peroxidase but not homocysteine, SOD and 
cystathionine. Significantly reduced activity of key GSH metabolism enzymes was 
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reported in cerebellum of individuals with autism including glutamate-cysteine ligase, 
modifier subunit (GCLM), GPX and glutathione transferases (GSTs), as well as 
paroxanase 1 (PON1), an enzyme that is inactivated by oxidized GSH. 
In addition to the previously mentioned relevant gene associations rare variants were 
reported in the 5' region of SOD1 in a few cases of autism.  Last, increases of 
mitochondrial DNA damage have been reported in autism and this is likely due to an 
increase in oxidative stress.  
Other important functions of GSH consist of S-glutathionylation of proteins (most likely 
serving as a protective measure against oxidative stress), reduction of glutaredoxin, 
reduction of ascorbate and tocopherol, direct removal of free radicals and protection of 
protein sulfhydryls. GSH may also be important in cell signaling through 
glutathionylation and, based on its action on NMDA receptors, may modulate glutamate 
signaling. It has also been reported that GSH may be an NMDA receptor agonist. It is 
therefore possible that GSH levels may contribute to the variability seen in the 
phenotypic response to sulforaphane treatment. It is interesting to note that the GSH 
system and specifically the GST's are important metabolizers of both the pesticides and 
air pollutants that have been suggested to be risk factors for autism. 
Significance: 
Currently, there are only two FDA-approved medications for ASD that we know of and 
they are approved for irritability associated with autism. None so far addresses the core 
features (e.g., social responsiveness). 
Sulforaphane is well tolerated and has shown negligible to no toxicity. Replication of the 
findings that sulforaphane may influence social interaction, abnormal behavior, and 
verbal communication in  autistic disorder will lead to a large scale, multi-center, clinical 
trial in adolescents and adults with autism as well as trials in younger individuals with 
autism. Promising results seen in several early intervention studies suggest that there is 
a timeframe in which some may be rescued from lifelong symptoms. It is therefore 
possible that along with early intervention protocols or by itself sulforaphane treatment 
may give a better response and maybe a long term improvement when applied to those 
with very early diagnosis of autism. 
Sulforaphane as a treatment for autistic disorder is intriguing since it intersects with 
several metabolites, systems and genes that we and others have previously implicated 
in autism. 
3. Research Design and Methods
This is a study comparing Avmacol® and placebo as adjuncts in the treatment of 
autistic disorder. Forty adolescent or adult outpatients total, with age ranges from 13 
to 30 years will be randomized into an 18-week (close out at 22 weeks) double-
blind, placebo-controlled parallel treatment study in a 2:1 treatment to placebo ratio. 
The study Dr. , Eveline Traeger, MD, is an expert in autism and will perform study 
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assessments including lab work, scales such as the Aberrant Behavioral Checklist 
(ABC) while remaining blind to medication regimens. Study assessments will be 
administered at designated time points. 
Research Design:
Subjects that express interest in the project will be called to see if they meet the 
inclusion and exclusion criteria. The studies Phone Script will be used for this call. 
Those that pass inclusion/exclusion and wish to be part of the project will undergo 
informed consent/assent. This will be done in a private location, preferably in the 
Staged Research Building room (SRB)115. Alternatives more convenient for the 
subject/family can be used as long as they are private. Consenting/assenting will be 
done by the Study Coordinator. When the study coordinator is not able to, the 
diagnostician or study doctor will perform informed consent/assent the subjects. 
NDAR GUID (National Database for Autism Research Global Unique Identifier Web 
Service, from the National Institutes of Health) will be used to generate ID numbers. 
Only GUID numbers will be used on documentation, tests and sample tubes. Only 
the screening questionnaire page 2 the Study Visit form and the NDAR GUID for, 
will link GUIDs to the subjects. These questionnaires will be stored in a locked file 
cabinet separate from other study information and only GUIDs will be used for 
electronic data.  The NDAR GUID helps to ensure that this information can be sent 
and stored securely, protecting the privacy of the individuals in the study and the 
confidentiality of their data. Using the NDAR GUID allows data collected on an 
individual from one study or site to be associated the data from another study or 
site without the risk of exposing PHI.  The identifying information used to generate 
the GUID remains on our secure computer and is used to generate “hashtags” 
which are used by NDAR to generate the GUID.  This unique ID number will be 
used to identify all samples, data and results. The Study Visit form will be kept by 
the study doctor in a locked cabinet/desk, in a secure office until the subjects has 
finished the study. The Study Visit form will then be destroyed.
Following informed consent/assent subjects will be tested with the studies 
diagnostic tools, ADOS-2 and DSM-IV. by one of the two study diagnosticians. Drs. 
Mars or Patel. These tests will be done in the SRB room 115. Those that are not 
eligible for the study will be informed of this and will no longer be part of the study. 
Study tests and documents will be stored securely in SRB room 107. 
Subjects that are diagnosed with moderate to severe autistic disorder will be invited 
to participate. Subjects will receive evaluations and medical tests by the study 
team, including the study doctor, Eveline Traeger, MD,  in either the Clinical 
Academic Building or the Clinical Research Center (CRC). This may include a 
clinical blood draw and urine collection to measure liver, thyroid and kidney function 
which will be done in the Clinical Research Center (CRC). It may include one 5ml 
draw for serum and one 5ml EDTA draw and 5ml of urine.  
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A checklist will be used for each subject visit. Additionally, two exam forms will be 
used for each visit. The first is to be used by the study Dr. called “Exam for study 
doctor” and the second to be used at the CRC called the “Exam form CRC”  will be 
used for each visit.
For those subjects that allow a blood draw,  2ml of the blood samples will be used 
for research and will be used to test biomarkers and as a source of DNA. 
Glutathione levels will be measured at baseline and weeks 18 and 22. Glutathione 
levels will be used to try to account for variability in response to sulforaphane. DNA 
will be used to genotype a specific group of genes of sulforaphane metabolism to 
try to account for variability in response to sulforaphane. A cheek swab will be used 
for collecting DNA for the subset of subjects that will not undergo a blood draw.
Baseline study metrics Clinical Global Impression (CGI), Aberrant Behavioral 
Checklist (ABC) and by the Social Responsiveness Scale (SRS) will be performed 
in the Clinical Academic Building Suite 6100 6th FL  or the CRC
The CRC pharmacist will randomize subjects in a 2:1 sulforaphane:placebo ratio. 
The medication or placebo is subsequently started. Enough study 
medication/placebo will be given until the next scheduled visit plus 1 week.
If any subjects are found to have clinical blood/urine test results outside normal 
limits, we will ask the subject to discontinue study medication. If after 2 weeks the 
lab values return to within the study’s acceptable limits and the subject wish to 
remain in the study and the study doctor agrees we will then ask the subjects to 
resume the study medication. Any adverse effects will be logged on the adverse 
effects form.
If the lab tests remain abnormal the subject will discontinue study medication 
permanently (though will continue to be followed-up in the study). 
Follow up visits with the study doctor will be at weeks 4, 10, 18 and 22 (please see 
outline below). In each week the study doctor will perform a screen and ask 
questions to see if there are any adverse effects. In each of these weeks the study 
doctor will perform the CGI, the ABC and the SRS. 
In weeks 4 and 10 additional study medication/placebo will be dispensed. 
At weeks 4 and 18 Examination by study doctor and query for adverse effects will 
be done and phlebotomy may be done for test of liver, thyroid and kidney function, 
Study medication/placebo will be completed in week 18 and some of the blood draw 
may be used to see if there are changes in biomarker levels. 
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The final visit will be in week 22. Examination for adverse effects and phlebotomy 
will be done for test of liver, thyroid and kidney function, Blood may be drawn for 
testing of glutathione levels.
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Schedule of Study Evaluations:
Time Line
Informed 
consent
/assent
Diagnosis, 
ADOS 
DSM
Study 
medication
Tests of  autistic 
disorder 
symptoms
Physical 
exam and 
possibly 
clinical 
blood/urine 
tests
Screening / 
questioning 
for any 
adverse 
effects
Sample 
for 
biomarke
analysis
Screening /Enrollment  
visit:
Y
Y
N/A
N/A
N/A
N/A
N/A
 
Baseline visit:
N/A
N/A
y
CGI, ABC and 
SRS.
y
N/A
y
4 week (follow-up) visit:
N/A
N/A
y
CGI, ABC and 
SRS
y
y
n
 
 
10 week (follow-up) visit:
N/A
N/A
y
CGI, ABC and 
SRS
n
y
n
 
 
18 week (treatment stop) 
visit:
N/A
N/A
n
CGI, ABC and 
SRS
y
y
y
 
 
22 week (final/closeout) 
visit:
N/A
N/A
n
CGI, ABC and 
SRS
y
y
y
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Methods:
Glutathione analysis:
Pre- and post-treatment plasma samples will be analyzed for total glutathione 
(tGSH) and reduced glutathione (GSH). While there are multiple commercially 
available kits available for these assays a general problem is lower sensitivity and 
susceptibility to interference from contaminants.  More recently HPCL and LC-
MSMS techniques have been developed.  The latter are preferable because they 
are not subject to contamination, are very specific and are sensitive.  Therefore we 
will do the analyses for GSH and GSSG by LC-MSMS for this study.  Dr. Stein will 
use the method of Squellerio et al (Squellerio, Caruso et al. 2012). We have the 
necessary equipment and do isotope dilution assays of biomolecules routinely in 
our laboratory. These assays will be completed by Dr. Peter Stein at Rowan – SOM 
in when the last samle is collected. All samples will be stored in SRB until all 
subjects have completed the study. Samples will then be sent to Rowan for analysis 
as a single batch to avoid possible confounding factors. Due to a Rowan 
requirement that we must have an IRB approval before he can apply for an 
approval at Rowan neither he nor Rowan are listed as part of this project in the 
initial application. Once we receive IRB approval we will give a copy of the approval 
letter to Rowan for their IRB review. Once approved or determined to be exempt / 
non-human research we will amend our protocol to add Dr. Stein and Rowan to our 
protocol. 
Genotyping:
DNA will be extracted at pre-treatment using established protocols. Genotyping of 
SNPs of alleles of genes of sulforaphane metabolism will be done by TaqMan 
assays as per the manufacturer's protocol. Genotyping of the GSTM1- and GSTT1- 
null alleles (whole gene deletions) will be done by a quantitative method to account 
for all three genotypes as opposed to standard methods that only account for null 
and not null. We will use qPCR TaqMan assays that we have previously used.
3.1. Duration of Study
This is a project. Each subject will be randomized into an 18-week double-treatment 
study with a closeout visit at 22 weeks. 
3.2 Study Sites
Steven G. Busyke PhD. .  PI / Principal Investigator and study sponsor.
Rutgers University 
Associate Research Professor of Statistics 
110 Frelinghuysen Road Room 559 Hill Center Bldg 
Department of Statistics 
Piscataway New Jersey, 08854 
 
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Study Coordinator – Data Steward: Edward S. Stenroos 
Department of Neurology
Rutgers-RWJMS
675 Hoes Lane The Staged Research Building Room 107.
Piscataway, NJ 08854 
Subject diagnosis will be done at;
Rutgers-RWJMS
675 Hoes Lane The Staged Research Building Room 115.
Piscataway, NJ 08854 
The study doctor will see the subjects at 
Rutgers RWJMS Department of Neurology
Clinical Academic Building 
125 Paterson Street, Suite 6100 6th FL
New Brunswick, NJ 08901
We will use the CRC pharmacy and labs for phlebotomy ;
RWJ East Tower, 8th floor
125 Paterson St., New Brunswick, NJ 08901
New Brunswick, NJ 08901
Biomarker testing will be done at Rowan-SOM by T. Peter Stein PhD Professor of 
Surgery. 
Please note, biomarker testing will not be done until the last sample is collected. 
Since Rowan University will not review Dr. Stein’s IRB application until Rutgers has 
approved the IRB protocol Dr. Stein will not initially be part of the project with respect 
to this IRB application. Once we have received an approval letter we will send a 
copy to Dr. Stein for his IRB application at Rowan. Once approved we will amend 
our protocol to add Dr. Stein and Rowan to the project. Text referring to Dr. Stein 
has been left in for clarity of study design and methods.
2 Medical Center Drive Science Center 1-106 
Stratford Camden New Jersey, 08084 
Department of Surgery 
Rowan - School of Osteopathic Medicine 
3.3 Sample Size Justification 
Forty child, adolescent or adult outpatients with autistic disorder will be randomized. 
This project is designed to confirm a previous study (Singh, Connors et al. 2014) 
with the goal of justifying a large scale clinical trial. This number is therefore based 
on both the design of the Singh study and cost constraints of this award. 
Power Analysis:
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Based on the observed standard deviations for change observed in Singh et al 
(2014), the designed sample size with 10% attrition, and two-tailed tests with α = 
0.05 as described above, we estimate that with a simple t-test the proposed study 
would have 80% power to detect differences in change scores as small as 20.4 and 
20.5 in ABC or SRS, respectively. The published standard errors of the mean in 
Singh et al (2014), however, from which the standard deviations were derived, were 
not derived in the mixed effects model; the paper indicates that model-derived 
estimates would be smaller, suggesting power to detect small differences in change 
scores. The observed, unadjusted change scores in Singh et al (2014) were 19.4 for 
ABC and 18.4 for SRS.
3.4 Subject Selection 
The inclusion and exclusion criteria of this study are dictated by the previously 
reported study of Sing et al (Singh, Connors et al. 2014). Additional variables are 
avoided with the hopes that replication will result in a large scale trial with a broad 
inclusion including females and individuals with autistic disorder of younger ages 
and possibly those with a diagnosis of autism spectrum disorder other than autism. 
3.4.1 Inclusion Criteria
o
Autistic disorder diagnosis. 
o
Age between 13-30 years.
o
Male gender.
3.4.2 Exclusion Criteria
o
Absence of a parent or legal guardian and consent/assent, 
o
Those that can not or will not complete all visits and adherence to study 
regimen. 
o
Seizure within 2 years of screening, 
o
History of chronic kidney, liver or thyroid disease.
o
Impaired renal function (serum creatinine> 1.2 mg/dl).
o
Impaired hepatic function (> 2x upper limit of normal).
o
Impaired thyroid function (TSH outside normal limits). 
o
Current infection or treatment with antibiotics.
o
Chronic medical disorder (e.g., cardiovascular disease, stroke or diabetes) 
or major surgery within 3 months prior to enrollment. 
o
Less than 13 years or more than 30 years of age.
o
Female gender.
o
A diagnosis of autism spectrum disorder other than autistic disorder, for 
example, Asperger, PDD-NOS etc.
4. Study Variables
4.1 Independent Variables or Interventions 
This is a double blind placebo-controlled treatment trial in autistic disorder. .
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4.1.1 Drug or Device Interventions 
Dose Rational:
Broccoli sprout extracts have previously been used as a delivery agent for the 
isothiocyanate sulforaphane and its precursor glucoraphanin, in clinical studies 
conducted in the US and China (Shapiro, Fahey et al. 2006, Fahey, Talalay et 
al. 2012, Fahey, Wehage et al. 2012, Egner, Chen et al. 2014, Singh, Connors 
et al. 2014).  Mean bioavailability of glucoraphanin preparations lacking active 
myrosinase is roughly 10% of dose, whereas when active myrosinase is 
included in the dose bioavailability increases to about 50%, with a concomitant 
reduction in within- and between-subject variability(Shapiro, Fahey et al. 2006, 
Fahey and Kensler 2007, Fahey, Talalay et al. 2012, Fahey, Wehage et al. 
2012) }. Using glucoraphanin rich broccoli seed powder containing active 
myrosinase will allow us to deliver sulforaphane in predictable doses in a 
delivery vehicle that is highly stable and thus unlikely to lose potency over time.  
Nutramax Laboratories, Inc. has had a broccoli extract supplement on the 
market that has been tested by independent labs and has been found to be of 
high quality. They have now manufactured a version of their supplement, 
modified to the studies specifications, (Avmacol®) and it will be send to us 
accompanied by appropriate placebo capsules, to be used in clinical trials.  This 
supplement will be provided to us in the form of tablets that will contain a 
mixture of broccoli seed powder with 30 mg of glucoraphanin (equivalent to 68 
µmoles of glucoraphanin) and enough active myrosinase enzyme to fully 
hydrolyse glucoraphanin to sulforaphane. About 50% of ingested glucoraphanin 
is recovered as sulforaphane metabolites in 24 hour urine collections(Ye, 
Dinkova-Kostova et al. 2002). Nutramax will send Avmacol® and the placebo to 
the CRC pharmacy in bulk (500 pill jars) once IRB approval has been obtained.  
All pills will be from a single batch. Avmacol® is stable at room temperature for 
up to two years and does not need special consideration. Once opened, pills 
will be kept in a refrigerator to avoid potential loss of sulforaphane 
bioavailability. If  expiration of the study medication  occurs, Nutramax will send 
a second batch. 
FDA:
We have prepared and submitted an IND application to the FDA for use of 
Avmacol® in autism. We received a “Study May Proceed “ letter from the FDA. 
IND 127543.
Dose:
We will use the same dose considerations as the Singh study which is based on  
100umol/day for average weight subject (set at 68kg / 150lbs). For this study 
we will administer 1.47umol/kg/day. 
Each pill has ~ 37umol GR/ tablet which produces  ~14.8umol bioavailable 
sulforaphane/ tablet (based on an observed 40% conversion efficiency). 
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Therefore; 
Body weight
Body weight
Dose of sulforaphane
45 kg
100 lb.
~ 50 µmol
3
68 kg
150 lb.
~ 100 µmol
6
102 kg
225 lb.
~ 150 µmol
9
Pill grinders will be supplied for those that have problems taking pills. The 
ground up pills can be added to food.
4.2 Dependent Variables or Outcome Measures
We will test for phenotypic change by the Clinical Global Impression (CGI), 
Aberrant Behavioral Checklist (ABC) and by the Social Responsiveness Scale 
(SRS) at baseline, week 4, week 10, week 18 and at a 22 week close out visit 
to assess phenotypic change in male individuals with autistic disorder receiving 
daily supplementation with Avmacol® .
Kaufman Brief Intelligence Test (KBIT):
All potential subjects will undergo the Kaufman Brief Intelligence Test (KBIT) to 
determine ability to undergo informed consent/assent. An IQ score greater than 
2 SD from mean (IQ score < 70) will be used as a cutoff point. This test will take 
15 – 30 minutes.
Diagnosis:
We will include male subjects with moderate to severe autistic disorder using 
the same two diagnostic criteria used in the Singh study, The Diagnostic and 
Statistical Manual of Mental Disorders, 4th Edition (DSM-IV), and The Autism 
Diagnostic Observation Schedule 2(ADOS-2). We will also score the more up to 
date tool DSM-5 for possible additional analysis after the study is complete.
Drs. Audrey Mars or Brenda Patel will assess phenotype of subjects with ADOS 
and DSM-IV and score DSM-5. Due to the inclusion criteria it is not likely that 
many potential subjects will have been recently diagnosed for other studies and 
will therefore need to be re-evaluated. If subjects have been recently diagnosed 
we may review these test results/records with the goal of relieving the burden 
on subjects and families. Drs. Mars and Patel will do so if they feel it is 
appropriate.  Administration time is approximately 45 minutes. 
Outcome Measures:
Outcome measures will be performed by Drs. Traeger and Nallapati. 
As in the Singh study the primary tests metrics will be the Aberrant Behavior 
Checklist (ABC) and Social Responsiveness Scale (SRS). A secondary metric 
will be the Clinical Global Impression Improvement Scale (CGI-I).
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Administration time is approximately 45 minutes.  
These tests are standard, proprietary tools or measures and are included in 
Appendix A., Some of these tests contain internal scoring (e.g. carbon paper) 
and were copied for review and are watermarked as “Sample”.
We will test for differences in the average change in ABC and SRS scores from 
baseline to 18 week, and their reversion to baseline at 22 week between those 
receiving sulforaphane and placebo.  As a secondary test we will look at the 
change in the CGI-I scores from baseline to each follow-up time point for each 
participant.
Administration time is approximately 45 minutes. 
Testing for possible treatment variability:
Biomarker: As previously mentioned we will be testing for differences in pre- 
and post-treatment glutathione levels in plasma samples and will measured for 
total glutathione (tGSH) and reduced glutathione (GSH). 
Genetic data: DNA will be extracted from the pre-treatment sample. Genotyping 
of SNPs and InDel alleles of genes of sulforaphane metabolism will be done 
genotyped. 
4.3 Chart Review Selection
Not applicable.
4.3.1 Electronic Medical Records
Not applicable.
4.4 Risks of Harm 
One risk to subjects is the potential side effects of sulforaphane. Sulforaphane 
is derived from eating cruciferous vegetables. The form used in this study is 
well tolerated and has shown no toxicity at the doses to be used in this study. 
We will carefully monitor subjects for adverse effects or clinical laboratory 
results outside of range. 
Possible clinical side effects of sulforaphane include: flatulence, weight gain, GI 
disturbances (large bowel movement, soft stool), and increased urination. 
There was no higher prevalence of these side effects in the treatment arm vs. 
placebo arm in the Singh study with the exception of weight gain (4.31 vs. 0.31, 
9 = 0.056 (Singh, Connors et al. 2014). 
These possible clinical side effects are relatively minor when compared to the 
possible improvement of core symptoms of autism.
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As with all studies there is a small risk of the loss of confidentiality.  All efforts 
will be made to keep personal information and research records confidential, 
but total confidentiality cannot be guaranteed. All data including screening data, 
physical results, blood test results, phenotypic data and test results will be 
securely sent to or brought to the Data manager/study coordinator. Data will be 
stored in both electronic files and hard copy source documents. The source 
documents will be coded and will be stored in locked file cabinets in a locked 
room. Any electronic data will be stored on password protected computers on a 
secure network within the University and not accessible by outside individuals. 
Sample collection tubes will not contain PHI. Each collection tube will be 
labeled with the subject’s unique ID number. Data analysis and reporting will be 
done with the Unique ID number only. 
These risks are relatively minor when compared to the possible improvement of 
core symptoms of autism. 
4.5 Potential for Benefit
Confirmation that sulforaphane treatment may influence social interaction, 
abnormal behavior, and verbal communication in male individuals with autistic 
disorder without serious side effects would be a major step in helping families 
and individuals with autistic disorder and may benefit the subject directly. Follow 
up studies will be done that may benefit females with autism as well younger 
individuals with autism. However, it is possible that subjects might receive no 
direct personal benefit from taking part in this study.
5.  Subject Recruitment and Enrollment Considerations
5.1
Subject Recruitment
We will attempt to give as many potential subjects the opportunity to enter this 
study as possible. Only IRB-approved recruitment tools will be used. As 
previously mentioned, we expect that few subjects will be verbal. We therefore 
expect that recruitment will be through parents or legal guardians.  Those 
subjects/parents/guardians that learn of our study and express interest will be 
contacted at their best convenience. Those that wish to enter the study will be 
scheduled for informed consent/assent. No information will be kept for those that 
decide they are not interested. Those that may be interested in future studies will 
be informed of the NJ ACE website which may help inform them of such studies.
Study patients will be recruited in several ways including local pediatric and 
pediatric neurology clinics, reference from other physicians, postings of study, 
invitation to those from previous studies that expressed a wish to be notified 
about future studies. Recruitment from local clinics, including pediatric, 
neurological and psychiatric. We will reach out to schools for individuals with 
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autism and other community groups serving the autism spectrum community as 
well. We will also use local advertising if necessary. In addition, we will have the 
benefits of the NJ ACE coordinating center at Montclair State University. We 
expect the coordinating center’s expertise and experience will be a significant 
asset in recruitment and completing this project.  Based on previous experience 
we expect to need to recruit about 70-100 subjects to have at least 40 subjects 
that pass inclusion/exclusion criteria, screening and complete the study. 
Additional recruitment tools will be sought out throughout the study and added if 
appropriate. 
Recruitment will be done by  the study coordinator. 
5.2 Consent Procedures
Subjects will be between 13 and 30 years old. . Determination of capacity to 
undergo informed consent/assent is crucial. All potential subjects, not clearly 
verbal will undergo the Kaufman Brief Intelligence Test (KBIT) to determine 
ability to undergo informed consent/assent. 
Any subjects that are clearly verbal will undergo informed consent if 18 or older 
and informed assent if between 13 and 17 years old prior to KBIT and 
diagnostic testing. Those that assent and turn 18 during the study will undergo 
informed consent at the next study visit. 
All subjects determined to be verbal and can understand and have the capacity 
will undergo informed consent/assent. 
Those that are not verbal and under 18 will be consented for by the parent / 
legal guardian.  
Those that are not verbal and over 18 and their parent / guardian has/have 
been granted legal guardianship will be consented for by the parent / legal 
guardian.
Those that are not verbal and over 18 and their parent / guardian has/have not 
yet been granted legal guardianship will be consented for using the surrogate 
consent process. An attending physician that is not part of/related to this project 
will assess ability to consent. A person, with no connection to this study will 
witness the surrogate consent process and sign the Surrogate Consent form.  
The person will be the chaplain at RWJ or someone identified prior to the need 
for consent that can attest that the requirements for informed consent to enter 
this study have been satisfied. If a subject expresses resistance or dissent they 
will be removed from the study. The subject will undergo informed consent if 
they become verbal and gain the ability to consent for themselves during the 
study.
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Subjects will be assessed for improvement at each study visit. Any non-verbal 
subjects over 18 that become verbal during treatment will undergo the Kaufman 
Brief Intelligence Test (KBIT) to assess ability to undergo informed consent. 
Those determined to have sufficient improvement and gain the capacity to 
undergo informed consent and that are literate will undergo informed consent.  
Those that have been consented for and turn 18 during the study and have 
gained the capacity to understand and undergo informed consent as described 
in the previous paragraph will undergo informed consent at the current or next 
scheduled visit. 
Any subjects that gain the capacity to undergo informed consent that dissent to 
participation in the study will be removed from the study as soon as safe 
termination can be affected.
The consent/assent will be made available to the subject/family by mail prior to 
the first appointment if desired, or at the time of the first appointment. 
Each subject and a parent / legal guardian will read through the consent/assent 
with the study member and have a dialogue about the study procedures, risks 
and potential benefits. Time will be provided, and if necessary, another 
appointment will be scheduled if a potential subject/parent/legal guardian 
requires further time for their decision or would like consultation with another 
individual prior to signing the consent/assent. All subject/parent/legal guardians 
will be reminded prior to signing a consent/assent that the study is voluntary, 
and they have the right to withdraw from the study at any time for any reason. 
All individuals providing consent/assent must be literate. Consenting/assenting 
will be done in a private location, preferably in the Staged Research Building 
room (SRB)115. Alternatives more convenient for the subject/family can be 
used as long as they are private. Consenting/assenting will be done by the 
Study Coordinator. When the study coordinator is not available/able to, the 
diagnostician or study doctor will perform informed consent/assent of the 
subjects.
Withdrawal: Subjects/parents/legal guardians that wish to withdraw from the 
study will be removed as soon as a safe termination can be affected. Any that 
wish to have data already collected destroyed/deleted must request this in 
writing.  NDAR will be informed by the PI to delete the subject’s data. Any data 
already submitted to other PIs by NDAR will not be retracted as per NDAR 
policy. Loss of confidentiality is not likely since no link will be present.
5.3 Subject Costs and Compensation
There is no cost to participate in the study other than travel expenses. There is 
a small reimbursement for travel to and from the research site. Each subject will 
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receive $5 for each scheduled visit. All study costs (e.g. study medication, 
tests, etc.) are covered by the grant from The Governor's Council for Medical 
Research and Treatment of Autism of New Jersey.
6.   Data Handling 
NDAR GUID (National Database for Autism Research Global Unique Identifier Web 
Service, from the National Institutes of Health) will be used to generate ID numbers. 
Only GUID numbers will be used on research documentation and tests from this 
point forward. Only the screening questionnaire page 2, the Study Visit form and 
the NDAR GUID from will link subject’s names to IDs. All other documents will 
contain only GUID numbers. These will be stored in locked file cabinet separate 
from other study information. Only GUIDs will be used for research related 
electronic data.  The NDAR GUID helps to ensure that this information can be sent 
and stored securely, protecting the privacy of the individuals in the study and the 
confidentiality of their data. Using the NDAR GUID allows data collected on an 
individual from one study or site to be associated the data from another study or 
site without the risk of exposing PHI.  The identifying information used to generate 
the ID is encrypted.  This unique ID number will be used to identify all samples, 
data and results. The Study Visit form will be kept by the study doctor in a locked 
cabinet/desk, in a secure office until the subjects has finished the study. The Study 
Visit form will then be destroyed.
Information needed to generate the NDAR GUID, including name as it appears on 
birth certificate, birthday, sex and town or municipality of birth as on the birth 
certificate will be kept on the “NDAR GUID form” and stored separately and 
securely from all other study documents in a locked file cabinet in room 107 of SRB. 
This form/data/link will be kept until data has successfully been uploaded to NDAR 
and then it will be destroyed. For those that do not wish their data to be uploaded to 
NDAR destruction of this form//data/link will occur immediately after the GUID has 
been generated successfully.
Only data from those that indicate on the consent/assent form that their data may 
be sent to NDAR will be uploaded to NDAR. 
All other forms, including screening questioners, consent/assent forms and 
diagnostic material will be kept in a locked file cabinet in room 107 of SRB. Only the 
study coordinator and PI will have access to these documents. Clinical documents 
and phenotypic tests will be brought to SRB room 107 the day of the visit when 
possible or stored in the CRC until the study coordinator can bring to SRB room 
107 and placed in the locked cabinets. Dr. Stein will receive sample with NDAR 
GUID's only. No PHI will be placed on sample tubes. Dr. Buyske the study 
statistician will not receive PHI. Study data including glutathione levels and 
genotypes will be stored in the project database which will be specific to this project 
and will not contain identifying information. The database will be password-
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protected on a password-protected computer in room 107. We have storage space 
on a University secured shared drive (Core) for storage. Subject and regulatory 
data will be kept for a minimum of 6 years. The link to identifying information used 
to generate the NDAR GUID will be destroyed as described above. Following this 
only the screening questionnaire and the Study Visit form will contain the link 
between name and GUID. Name and contact information will be part of the subject 
records and will be kept for a minimum of 6 years. Screening questionnaires for 
those that do not enter the study will be destroyed.
7.
Statistical Analysis
Our statistical methods mirror those of the original Singh study. There is an unfortunate 
tendency, known as "researcher degrees of freedom" or (less generously) "p-hacking" 
by which investigators, perhaps subconsciously, make analysis decisions that favor 
significant results. This process has been proposed as a partial mechanism for the 
phenomenon of studies that do not replicate.
Since the proposed study is a replication of an existing study, we plan on a statistical 
analysis that fully matches the analysis in the original study. In particular, for Aim 1, we 
will use as our primary outcome the change in Aberrant Behavior Checklist (ABC) and 
Social Responsiveness Scale (SRS) scores from baseline to 18 weeks and the change 
from 18 to 22 weeks. Changes in Clinical Global Impression Improvement Scale (CGI-I) 
scores, as well as changes in all scores from baseline to other time points, will be 
treated as secondary analyses. We will use a mixed effects general linear model, with 
fixed effects for both week and the interaction of post randomization week and 
treatment group and random effects for intercept and slope of participants. Unstructured 
covariance will be used. As shown in (Liang KY 2000), the shared baseline implied by 
the lack of a main effect for treatment corresponds to the undifferentiated (with respect 
to treatment) population before randomization and reduces variance much as adjusting 
for baseline does in ANCOVA. The mixed effects model allows for all participants with at 
least one post randomization visit to be included in the analysis. An intent-to-treat 
analysis will be done to supplement the per-protocol analysis.
Test statistics will be based on contrasts corresponding to differences between 
treatments in the baseline-to-visit scores, except for the test of behavior regression, 
which will be based on a contrast from week 18 to week 22. We will use two-tailed tests 
with alpha = 0.05, both to match the original study and to follow the usual practice of a 
study replicating an established result.
Power Analysis:
We plan on recruiting sufficient amount of subjects to have 40 participants finish the 
trial. That said based on the same power calculation assumptions in Singh et al. (2014), 
the designed sample size with 40 participants, 10% attrition, and two-tailed tests with 
alpha = 0.05 as described above, we estimate that with a simple t-test the proposed 
study would have 80% power to detect differences in change scores as small as 16.3 
for ABC or SRS. The more refined mixed effects model should have even greater power 
(and so the ability to detect significance for smaller change score differences). For 
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context, the observed change scores in Singh et al. (2014) were 19.4 for ABC and 18.4 
for SRS. 
8.
Data and Safety Monitoring 
While this study does not put the subjects at greater than minimal risk we will 
assemble a Data and Safety Monitoring Board. 
The Data and Safety Monitoring Board will be composed of no less than three 
persons with one being a physician (MD) familiar with treatment of autism and a 
statistician, none with any affiliation to the study.  The monitoring board shall 
determine safe and effective conduct and recommend conclusion of a trial if 
significant risks develop or the trial is unlikely to be concluded successfully.  The 
DSMB will conduct an open session prior to the initiation of the trial.  The DSMB will 
have been provided all material (protocol, consents, and procedures) associated 
with the trial, and have had sufficient opportunity to review the documents.  
Specifically, the Data and Safety Monitoring Board shall be responsible for:

Reviewing the research protocol and plans for data and safety monitoring.

Monitoring that will take place on a regular basis: every quarter.  

Evaluating the progress of the trial, including periodic assessments of data 
quality and timeliness, participant recruitment, accrual and retention, participant 
risk versus benefit, and other factors that may affect study outcome.  Monitoring 
may also consider factors external to the study when interpreting the data, such 
as scientific or therapeutic developments that may have an impact on the safety 
of the participants or the ethics of the study.

Inquiring for further information as necessary to accomplish their mission.

Maintaining confidentiality during all phases of the trial including the monitoring, 
preparation of interim results, review and response to monitoring 
recommendation.  

Generating a report that will be provided to the PI and the IRB.
The Principal Investigator of the trial, Steve Buyske, shall be responsible for:

Evaluation of any members of the DSMB for a conflict of interest or financial 
stake in the outcome of the trial.

Delegation of the ongoing monitoring of the trial to the DSMB.

Ensuring that monitoring is timely and effective and the DSMB is composed of 
individuals with appropriate expertise to accomplish their tasks.

Overseeing the monitoring activities.

Responding to and with the study doctor, addressing recommendations that 
result from monitoring activities.

Provision of adverse event reports and other safety data to the DSMB, as well as 
any changes in the trial or annual reports to the IRB and DOD.

Contributing to the report that the DSMB generates, which will be distributed to 
the IRB.

Submitting the DSMB report to the IRB.
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Protocol deviations:
Protocol deviations will be reported to the IRB, the DSMB as well as the funding 
institutions as required. 
Withdrawal from the Protocol:
All the individuals and parents/guardians will be reminded at the time they sign 
consent/assent that the study is voluntary and they have the right to withdraw from the 
study at any time for any reason. If they wish for samples or data to be withdrawn we 
will request they inform us of their decision, in writing. NDAR will be informed by the PI 
to delete the subject’s data. Any data already submitted to other PIs by NDAR will not 
be retracted as per NDAR policy. Loss of confidentiality is not likely since no link will be 
present.
9. Reporting Results
9.1 Individual Results
The study physician will report abnormal clinical test results received from the CRC 
with the subject/parents/guardian as soon as received and they will be asked to stop 
taking the study medicine/placebo immediately. 
9.2 Aggregate Results
Results of the study will be made available for individuals in the study once results 
have been analyzed and published. Individuals will not be identified in any of the 
study publications. Details about study medication and response will be made 
available to those subjects/families to those that request after data has been 
analyzed. 
9.3Professional Reporting
Results of this study will be published in peer review journals. Presentations will be 
made at appropriate seminars and conferences including appropriate conferences 
held by the Coordinating Center of Montclair State University (CC-MSU) – NJ Autism 
Center of Excellence. No identifying information will be used in publications. 
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H. Lambert and W. G. Johnson (2007). "Risk of autistic disorder in affected offspring of mothers 
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