AN OPEN-LABEL, ACUTE CLINICAL TRIAL TO ASSESS THE LEVEL OF KETONE PRODUCTION FOLLOWING CONSUMPTION OF AVELA™ (R)-1,3-BUTANEDIOL IN AN ADULT POPULATION
Phase: NA
Conditions: Healthy
Sponsor: Genomatica Inc.

 
AN OPEN-LABEL, ACUTE CLINICAL TRIAL TO ASSESS THE 
LEVEL OF KETONE PRODUCTION FOLLOWING 
CONSUMPTION OF AVELA™ (R)-1,3-BUTANEDIOL IN AN 
ADULT POPULATION 
Principal Investigator  
Lowder, James Norman, M.D.  
James Lowder Consulting LLC 
132 Barn Owl Ct 
Gore, VA 22637 
Funding Sponsor 
 
Genomatica Inc. 
4757 Nexus Center Dr. 
San Diego, CA 
9212, U.S.  
Study Product 
 
Avela™ (R)-1,3-Butanediol 
Protocol Number 
 
IRB Number 
CHSI-2101-02  
22-GMAT-101. 
 
Initial version 
22 March 2022 
Amended 
11 May 2022 
 
 

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Table of Contents 
BACKGROUND AND STUDY RATIONALE ............................................................................................... 1 
1 
INTRODUCTION ................................................................................................................................. 1 
1.1 
BACKGROUND ............................................................................................................................... 1 
1.2 
STUDY PRODUCT .......................................................................................................................... 1 
1.3 
GENERALLY RECOGNIZED AS SAFE (GRAS) FOR USE .................................................................... 1 
1.4 
CLINICAL DATA ............................................................................................................................. 2 
1.5 
CONCLUSIONS .............................................................................................................................. 4 
1.6 
DOSE RATIONALE AND RISK/BENEFITS ........................................................................................... 4 
2 
STUDY OBJECTIVES ........................................................................................................................ 4 
2.1 
PRIMARY OBJECTIVE ..................................................................................................................... 4 
2.2 
SECONDARY OBJECTIVES (IF APPLICABLE) ..................................................................................... 4 
3 
STUDY DESIGN ................................................................................................................................. 4 
3.1 
GENERAL DESIGN ......................................................................................................................... 4 
3.2 
STUDY ENDPOINTS ....................................................................................................................... 5 
3.2.1 
Primary Study Endpoint ......................................................................................................... 5 
3.2.2 
Secondary Study Endpoints ................................................................................................... 5 
3.3 
PRIMARY SAFETY ENDPOINTS ....................................................................................................... 5 
4 
STUDY POPULATION AND DURATION OF PARTICIPATION ........................................................ 5 
4.1 
DURATION OF STUDY PARTICIPATION ............................................................................................. 5 
4.2 
TOTAL NUMBER OF SUBJECTS AND SITES ....................................................................................... 5 
4.3 
INCLUSION CRITERIA ..................................................................................................................... 6 
4.4 
EXCLUSION CRITERIA .................................................................................................................... 6 
4.5 
SUBJECT RECRUITMENT ................................................................................................................ 6 
4.6 
EARLY WITHDRAWAL OF SUBJECTS ............................................................................................... 7 
4.6.1 
When and How to Withdraw Subjects .................................................................................... 7 
4.6.2 
Data Collection and Follow-up for Withdrawn Subjects ......................................................... 7 
5 
STUDY PRODUCT ............................................................................................................................. 7 
5.1 
DESCRIPTION AND TREATMENT REGIMEN ....................................................................................... 7 
5.2 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ......................................................... 8 
5.3 
SUBJECT COMPLIANCE MONITORING .............................................................................................. 8 
5.4 
PRIOR AND CONCOMITANT THERAPY .............................................................................................. 8 
5.5 
HANDLING OF STUDY PRODUCT ..................................................................................................... 8 
5.5.1 
Dispensing of Study Product .................................................................................................. 8 
5.5.2 
Return or Destruction of Study Product ................................................................................. 9 
6 
STUDY SCHEDULE ........................................................................................................................... 9 
6.1 
SCREENING – DAY 1 (INVESTIGATIVE STAFF) ................................................................................ 10 
6.2 
STUDY TEST DAY – DAY 2 (STUDY PARTICIPANT) ......................................................................... 10 
6.2.1 
Prior to Study Start ............................................................................................................... 10 
6.2.2 
0 min timepoint ..................................................................................................................... 10 
6.2.3 
30 min timepoint ................................................................................................................... 11 
6.2.4 
60 min timepoint ................................................................................................................... 11 
6.2.5 
90 min timepoint ................................................................................................................... 11 
6.2.6 
120 min timepoint ................................................................................................................. 11 
6.2.7 
180 min timepoint ................................................................................................................. 11 
6.2.8 
240 min timepoint ................................................................................................................. 11 
6.2.9 
300 min timepoint ................................................................................................................. 11 

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6.3.0 
End of Study ......................................................................................................................... 12 
7 
STUDY PROCEDURES .................................................................................................................... 12 
7.1 
CLINICAL EVALUATIONS ............................................................................................................... 12 
7.2 
LABORATORY EVALUATIONS ........................................................................................................ 12 
7.2.1 
Clinical Laboratory Evaluations: Blood BHB Measurement ................................................. 12 
7.3 
QUESTIONNAIRE ASSESSMENTS .................................................................................................. 13 
7.3.1 
GI Symptoms Assessment ................................................................................................... 13 
7.3.2 
Assessment of Alertness ...................................................................................................... 13 
8 
STATISTICAL ANALYSIS PLAN ..................................................................................................... 13 
9 
SAFETY AND ADVERSE EVENTS ................................................................................................. 13 
9.1 
DEFINITIONS ............................................................................................................................... 13 
9.2 
RECORDING OF ADVERSE EVENTS ............................................................................................... 14 
9.3 
REPORTING OF SERIOUS AES AND UNANTICIPATED PROBLEMS ..................................................... 14 
9.3.1 
Investigator reporting: Notifying the IRB .............................................................................. 15 
10 
STUDY ADMINISTRATION, DATA HANDLING AND RECORD KEEPING ................................... 15 
10.1 
CONFIDENTIALITY ........................................................................................................................ 15 
10.2 
SOURCE DOCUMENTS ................................................................................................................. 15 
10.3 
RECORDS RETENTION ................................................................................................................. 15 
11 
STUDY MONITORING, AUDITING, AND INSPECTING ................................................................. 16 
11.1 
STUDY MONITORING PLANS ......................................................................................................... 16 
11.2 
AUDITING AND INSPECTING .......................................................................................................... 16 
12 
ETHICAL CONSIDERATIONS ......................................................................................................... 16 
13 
STUDY FINANCES ........................................................................................................................... 16 
13.1 
FUNDING SOURCE ...................................................................................................................... 16 
13.2 
SUBJECT STIPENDS OR PAYMENTS .............................................................................................. 16 
14 
PUBLICATION PLAN ....................................................................................................................... 16 
15 
REFERENCES .................................................................................................................................. 17 
16 
APPENDICES ................................................................................................................................... 18 
APPENDIX A 
SCREEN CAPTURES OF ONLINE SCREENING SURVEY ....................................................... 19 
APPENDIX B 
DETAILED INSTRUCTIONS FOR PARTICIPANTS .................................................................. 20 
B-1 Detailed instructions for participants ........................................................................................... 20 
APPENDIX C 
SCREEN CAPTURES OF ONLINE PORTAL ............................................................................ 1 
 
LIST OF FIGURES AND TABLES 
Table 3.1-1 
Study Groups Included in Clinical Study ............................................................................ 5 
Table 5.1-1 
Clinical Study Outline ......................................................................................................... 8 
Table 6-1 
     Order of Events on Study Test Day ................................................................................. 10 
 

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List of Abbreviations 
AcAc 
acetoacetate 
AE 
adverse event 
ATP 
Adenosine triphosphate 
BHB 
β-hydroxybutyrate 
BMI 
body mass index 
EC 
Ethics Committee 
GI 
gastrointestinal 
GRAS 
Generally Recognized as Safe 
HIPPA 
Health Insurance Portability and Accountability Act of 1996 
IRB 
Institutional Review Board 
mVAS 
modified visual analogue scale 
n 
number 
PHI 
protected health information 
PI 
Principal Investigator 
SAE 
serious adverse event 
SSS 
Stanford Sleepiness Scale 
U.S. 
United States 
VO2 
Peak oxygen uptake 
 
 

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Study Summary 
 
Title 
An Open-Label, Acute Clinical Trial to Assess the Level of Ketone Production 
Following Consumption of Avela™ (R)-1,3-Butanediol in an Adult Population  
Short Title 
Effects of (R)-1,3-butanediol on ketone production  
IRB Number 
  
Protocol Number 
CHSI-2101-02  
Methodology 
Open-label, Acute (single day) study 
Study Duration 
1 day for screening, 1 day for clinical study 
Study Center(s) 
Single-center  
Objectives 
Primary: 
• 
To evaluate the level of production of BHB following the consumption of 
3 servings of Avela™, with consumption of each serving separated by 
30 minutes and each serving providing 11.5 g of (R)-1,3-butanediol 
[total intake of 34.5 g of (R)-1,3-butanediol].  
Secondary: 
• 
To evaluate the acute effects of 3 servings of Avela™, with 
consumption of each serving separated by 30 minutes and each 
serving providing 11.5 g of (R)-1,3-butanediol [i.e., total intake of 34.5 g 
(R)-1,3-butanediol] on: 
o 
GI tolerance 
o 
Sleepiness/alertness 
Number of Subjects 
N = 30 subjects (males and females) 

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Main Inclusion and 
Exclusion Criteria 
Inclusion Criteria: 
• 
Males and females, 18 to 65 years of age 
• 
BMI 18 to <35.0 kg/m2, inclusive 
• 
Weight ≥ 110 lbs 
• 
Subject is judged to be in good health on the basis of medical history 
• 
Subject is willing to fast for 12 hours prior to study start 
• 
Subject is willing to avoid alcohol and intense physical activity the day 
prior to and on the day of testing 
• 
Subject is capable of giving informed consent and complying with all 
study procedures/requirements. 
Exclusion Criteria: 
• 
Previous GI disorders (e.g., inflammatory bowel disease, irritable bowel 
syndrome, history of surgery for weight loss, or gastroparesis) 
• 
Gastroenteritis in the 2 weeks preceding the study 
• 
Diabetes 
• 
Weight <110 lbs 
• 
History of alcohol or drug abuse 
• 
Previous diagnosis of neurological disorders, depression, or mental 
illness with psychosis 
• 
Unexplained alarm features (i.e., unintentional weight loss >10% body 
weight in the last 3 months, fevers, or blood in stools) 
• 
Use of an antibiotic or any medication impacting gut transit during the 2 
weeks before the study 
• 
Constipation or diarrhea (defined as, on average, less than 3 stools per 
week or more than 3 stools per day, respectively) 
• 
Allergy to tree nuts 
• 
Women who are pregnant or breastfeeding 
• 
Persons with medical conditions affecting the pancreas, liver, thyroid, 
or gall bladder. 
Study Product, Dose 
and Route of 
Administration 
Avela™, providing 11.5 g of (R)-1,3-butanediol per serving, 3 servings during 
the test day, with consumption of each separated by 30 minutes, for a total 
intake of 34.5 g (R)-1,3-butanediol. 
Statistical Methodology 
Statistical analysis plan to be completed by data management team. 
Data and Safety 
Monitoring Plan  
The PI will be responsible for data quality management and for overseeing 
clinical trial participant safety.   
BMI = body mass index; GI = gastrointestinal; N = number; PI = Principal Investigator; IRB = Institutional Review 
Board. 

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Background and Study Rationale 
1 
Introduction 
This document is a protocol for a pilot clinical research study.   
1.1 
Background 
The purpose of the pilot clinical trial described herein is to determine the level of ketone production 
[measured as β-hydroxybutyrate (BHB)] following consumption of (R)-1,3-butanediol in a beverage and to 
record gastrointestinal (GI) symptomology, as well as any effects on alertness/sleepiness.   
 
There are two distinct stereoisomers of 1,3-butanediol: the R form and the S form.  The R stereoisomer of 
1,3-butanediol is naturally occurring, has been well studied, and appears to be the configuration that can 
be converted to physiological ketone bodies most proficiently (Desrochers et al., 1992).  Both the R or S 
stereoisomers of 1,3-butanediol undergo interconversion via the enzyme BHB dehydrogenase, producing 
the metabolites BHB and acetoacetate (AcAc).  In the liver and blood, BHB is converted to other ketones, 
such as AcAc and acetone; this is similar to the endogenous pathway of AcAc formation in the liver from 
fatty acids (Shivva et al., 2016; Poff et al., 2019).  Circulating ketones enter other tissues including the 
brain, kidney, skeletal muscle, and heart, and act as energy sources generating adenosine triphosphate 
(ATP) via the citric acid cycle.  Excess circulating ketones that are not taken up into the tissues are 
eliminated in the form of acetone via exhalation through the lungs, or BHB and AcAc via the kidneys 
(Shivva et al., 2016; Poff et al., 2019).  Under normal physiological conditions, blood levels of ketones are 
<3 mg/100 mL and urinary levels of ketones are ≤125 mg/24-hour; however, during periods of limited 
glucose availability, humans increase ketone production to approximately 150 g of ketone bodies per day 
(Reichard et al., 1974).  During fasting conditions, blood BHB levels as high as 5 mM (42 to 52 mg/100 
mL) have been observed (Cahill, 1970). 
 
For over a century, the classic ketogenic diet has been used to induce ketosis; the ketogenic diet is a low-
carbohydrate, high fat diet or a low-calorie diet (Avgerinos et al., 2020).  More recently, ketogenic diets 
have proved popular among individuals seeking to achieve weight loss (e.g., the “Atkins diet”); these diets 
differ markedly from the classic ketogenic diet in that protein consumption is generally not restricted.  With 
excessive protein consumption, amino acids undergo conversion to glucose, and as such, diets such as 
the Atkins diet that do not restrict protein intakes are far less ketogenic than the traditional “classic” 
ketogenic diet.   
 
This study aims to evaluate the level of BHB production when Avela™ (R)-1,3-butanediol is consumed at 
a level of 34.5 g within a single test day.  
1.2 
Study Product 
Avela™ (R)-1,3-butanediol will be consumed in the form of 3 servings of a beverage, with consumption of 
each serving separated by 30 minutes and each serving providing 11.5 g of (R)-1,3-butanediol [for a total 
intake of 34.5 g (R)-1,3-butanediol during the test day].  
1.3 
Generally Recognized as Safe (GRAS) for Use 
Avela™ is manufactured by microbial fermentation using a strain of Escherichia coli that has been 
genetically engineered to produce (R)-1,3-butanediol.  The isolation of (R)-1,3-butanediol from the 
production organism and fermentation media is accomplished using a series of purification and filtration 
steps that yield a highly purified product [≥99.7% (R)-1,3-butanediol].  Avela™ (R)-1,3-butanediol is 
intended to induce ketosis in the general adult population.  Avela™ (R)-1,3-butanediol is GRAS for use in 
beverages, bars, and gels at a level of up to 11.5 g/serving and for 1 to 3 servings per day, yielding total 
intakes ranging from 11.5 to 34.5 g (R)-1,3-butanediol/day (equivalent to 0.16 to 0.49 g/kg body 
weight/day).  In the pilot clinical trial described herein, study participants will consume a beverage 3 times 
during the test day, with consumption of each serving separated by 30 minutes and each serving 
providing 11.5 g of (R)-1,3-butanediol, for a total (R)-1,3-butanediol intake of 34.5 g on the test day; thus, 

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use of (R)-1,3-butanediol in the clinical trial described herein is within the parameters of the GRAS uses 
and use levels.      
1.4 
Clinical Data 
There were 5 clinical studies (Kies et al., 1973; Tobin et al., 1975 (Study 1 and 2); Scott et al., 2019; 
Shaw et al., 2019) in which the efficacy and/or safety/tolerability of (R, S)-1,3-butanediol were assessed.   
 
Kies et al. (1973) conducted a 4-arm crossover study in 12 healthy human volunteers [young adults, 18 to 
23 years of age; number (n)=6/sex] to evaluate the protein nutriture provided by nitrogen±urea when 
consumed with isocaloric quantities of either 1,3 butanediol (chirality not reported but assumed to be R,S) 
or starch.  Following a nitrogen depletion phase, the participants each partook in 4 arms of the trial.  
Wheat flour bread was formulated as follows for each of the 4 treatment arms: 1) Starch control 
supplemented with a variety of essential nutrients and minerals; 2) 5% w/w 1,3-butanediol (15 g, 
90 kcal/subject/day, substituted isocalorically for starch); 3) Starch plus urea (increased nitrogen content 
by 4 g/subject/day); 4) 5% w/w 1,3-butanediol plus urea (increased nitrogen content by 4 g/subject/day).  
Participants completed each 7-day treatment arm in an order that ensured 14 consecutive days of 
1,3-butanediol consumption and 14 consecutive days without, e.g., 2-4-1-3 or 3-1-4-2.  Samples of 
excrement were taken at each excretion during all arms, 7-day composites were made for each study 
arm, and venous blood samples were collected at study initiation at the end of each study arm, and 
2 weeks after study conclusion.  1,3-butanediol, with or without urea, did not significantly impact serum 
protein, blood cell counts (i.e., white blood cells, monocytes, eosinophils, lymphocytes, neutrophils), 
hematocrit, or hemoglobin levels in participants compared to treatment with starch alone.  No significant 
difference in the blood lipid or plasma ion content was reported following 1,3-butanediol consumption 
compared to baseline values.  The authors reported a significantly increased nitrogen balance in 
participants when consuming 1,3-butanediol alone compared to the starch control (p<0.05), accompanied 
by a significant decrease in urinary nitrogen excretion (p<0.05).  After consuming 1,3-butanediol alone 
(arm 2), the authors also reported a significant decrease in blood glucose when compared to all other 
treatment arms (p<0.05) (Kies et al., 1973).  1,3-butanediol was found to be a nontoxic metabolite 
providing calories for human nutrition and did not affect safety parameters, assessed using bloodwork, 
any differently than starch consumption.  
    
Tobin et al. (1975) conducted 2 studies; one to assess the effects of 1,3-butanediol (chirality not reported) 
on hematological measures in a fasting state and a second to assess the effects of prior consumption of 
1,3-butanediol on serum insulin, growth hormone, glucose and lipids during glucose tolerance tests.  The 
first study was a 2-arm crossover study that included a 5-day adjustment period (dietary nitrogen was 
supplemented with wheat protein) and 2 study arms of 5 days each, for a total of 15 days.  Following the 
adjustment period, study subjects (n=27 adult females) consumed for 5 days: 1) a diet containing 40 g of 
1,3-butanediol (chirality not reported), or 2) a control diet containing 40 g of sucrose.  Fasting blood 
samples were taken before and after each study arm for the analysis of hematological metrics (e.g., 
glucose, insulin, growth hormone, cholesterol, and triglycerides).  Blood glucose concentrations in the 
adjustment period were slightly higher than those reported in the 1,3-butanediol or control arms of the 
study, although not statistically different.  There were no other changes measured in the blood samples 
between the 2 study arms, but the authors did report a “significant” decrease (no statistics provided) in 
cholesterol during the treatment phases (both 1,3-butanediol and control) as compared to the adjustment 
phase, and the opposite was observed for blood triglycerides.  The authors concluded that there was “no 
evidence of toxic reactions to the ingestion of [1,3-butanediol] … detected” when administered at 40 g/day 
(Tobin et al., 1975).    
 
The second study by Tobin et al. (1975) was a 2-arm crossover study that evaluated the impact of 1,3-
butanediol (chirality not reported) on glucose-tolerance.  In a 12-day study, 10 healthy, young-adult 
subjects (male & female university students) were fed a controlled diet composed of an amino acid 
supplemented gelatin (6 g/nitrogen/day) and starch bread as primary energy sources.  Caloric intake for 
each subject was determined and 10% of their intake was isocalorically substituted for 1,3-butanediol or 
sucrose (control).  Subjects ingested either the 1,3-butanediol diet or sucrose diet for 5 days, and glucose 
tolerance tests were conducted on Day 6 following a 10 to 14 hour fast.  Study participants then 

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consumed the alternate diet for 5 days (Days 7 to 11), and glucose tolerance tests were conducted on 
Day 12 following a 10 to 14 hour fast.  There were no significant changes observed in any of the metrics 
tested compared to that of the control group including glucose, insulin, lactate, pyruvate, or triglycerides.  
Based on these findings, the authors suggest that 1,3-butanediol can be supplemented in the diet up to 
10% without risk of adverse effects (Tobin et al., 1975). 
 
Lastly, in the 2 clinical studies conducted by Scott et al. (2019) and Shaw et al. (2019), the effects of 
1,3-butanediol on exercise performance were assessed; in the study by Scott et al. (2019), chirality is 
unknown and in the study by Shaw et al. (2019), (R, S)-1,3-butanediol was used, but the proportional 
distribution of the stereoisomers is unknown.  Both studies were randomized, placebo-controlled, and 
crossover in design, with a 1-day intervention period.  A washout period of 7 to 10 days was included in 
the study by Shaw et al. (2019), whereas the duration of the washout period was not reported in the study 
by Scott et al. (2019).  One study was double-blind (Scott et al., 2019), whereas the other study was 
single-blind (Shaw et al., 2019).  In both studies, only male subjects were included, and mean body mass 
index (BMI) and peak oxygen uptake (VO2 peak) at baseline were similar across the 2 studies.  Scott et 
al. (2019) described the eleven subjects as healthy runners and did not provide further details on their 
athletic status, while Shaw et al. (2019) described the 9 subjects as trained cyclists, amongst whom the 
average amount of time spent training was 12.3 ± 2.3 hours/week and the mean maximal power output 
was 389.3 ± 50.4 W.  In the study by Scott et al. (2019), subjects first completed a 60-minute submaximal 
run, followed by a 5-km run time trial.  In the study by Shaw et al. (2019), subjects first completed an 85-
minute steady-state cycling period, followed by a cycling time trial to achieve 7 kJ/kg (which was reported 
to be approximately 25 to 35 minutes of cycling).  In both studies, 1,3-butanediol was consumed as part 
of a beverage.  In the study by Scott et al. (2019), the dose of 1,3-butanediol was 0.5 g/kg body weight 
and provided via approximately 650 mL of the test drink, which was consumed in 3 divided doses (twice 
before and once after the pre-time trial exercise).  In the study by Shaw et al. (2019), the dose of 1,3-
butanediol was 0.35 g/kg body weight and provided via approximately 278 mL of the test drink, which was 
consumed in 2 divided doses (once before and once during the pre-time trial exercise).  Across the 2 
studies, outcome measures related to exercise performance, GI tolerability, and hydration status, as well 
as blood biomarkers (i.e., BHB, glucose, and lactate), were assessed.  
 
Across both studies, there were no significant differences between the 1,3-butanediol and placebo 
phases on the time to complete the time trial, and the magnitudes of the effect sizes observed were 
interpreted as “unclear”.  Similarly, no significant differences between phases were observed for power 
output (Shaw et al., 2019) and pacing (Scott et al., 2019) during the time trial; the magnitudes of the 
effects were interpreted as “unclear” or “very likely trivial” (for pacing for the first 3-km of the 5-km time 
trial).  Thus, the consumption of 1,3-butanediol compared to placebo did not result in statistically 
significant or clinically significant improvements in time trial performance.  Additionally, the consumption 
of 1,3-butanediol compared to placebo significantly increased blood BHB levels throughout exercise.  
However, the consumption of 1,3-butanediol compared to placebo did not result in statistically or clinically 
significant, sustained improvements in supportive outcomes of exercise performance.  Furthermore, the 
evidence to support an effect of 1,3-butanediol on the sparing of glucose reserves is unclear.  It should be 
noted that, in the study by Scott et al. (2019), BHB was elevated following administration of 1,3-butanediol 
but was not sufficient to induce moderate or high levels of ketosis.   
 
In the study by Shaw et al. (2019), there was no difference in the change of body mass (corrected for fluid 
intake) between trials.  Within the group consuming butanediol, two participants experienced low levels of 
nausea, euphoria, dizziness.  Five participants reported low to moderate levels of belching and burping, 
and one participant reported severe abdominal pain.  All participants reported that they disliked the taste 
of butanediol, which resulted in retching in four participants.  In the study by Scott et al. (2019), no 
adverse GI effects were reported.   
 
It should be noted that, in the study by Shaw et al. (2019), a pilot test was conducted prior to the primary 
study to determine a testing dose of 1,3-butanediol in a beverage that was well tolerated and would 
provide optimal BHB blood concentration.  In the pilot test, single bolus doses of 0.5 g/kg and 0.7 g/kg of 
1,3-butanediol resulted in nausea, euphoria, and dizziness.  To resolve these issues, the authors 
determined a split dose of 0.35 g/kg (providing about 24.5 g/dose for a 70-kg individual) at the initiation of 

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the cycling trial and another at the midway point, which resulted in 0.7 g/kg 1,3-butanediol (49 g/trial for a 
70-kg individual), elicited maximal BHB concentration and minimal side effects. 
1.5 
Conclusions  
Avela™ (R)-1,3-butanediol is GRAS for use in beverages, bars, and gels at a level of up to 11.5 g/serving 
and for 1 to 3 servings per day, yielding total intakes ranging from 11.5 to 34.5 g (R)-1,3-butanediol/day 
(equivalent to 0.16 to 0.49 g/kg body weight/day).  In the clinical trial described herein, study participants 
will consume a beverage 3 times during the test day, with each serving providing 11.5 g of (R)-1,3-
butanediol, for a total (R)-1,3-butanediol intake of 34.5 g on the test day; thus, use of (R)-1,3-butanediol in 
the clinical trial described herein is within the parameters of the GRAS uses and use levels. 
Studies in humans demonstrated no significant adverse effects when 1,3-butanediol provided up to 10% 
of energy intake for 5 days (Tobin et al., 1975).  More recent studies reported that 3 doses up to 35 g/day 
(Scott et al., 2019) and split-dosing up to 49 g/day (Shaw et al., 2019) of (R, S)-1,3-butanediol (racemic 
mixture) were well-tolerated by healthy athletes.  Consumption of a single bolus dose of 35 or 49 g 
“tended to result in nausea, euphoria, and dizziness” (Shaw et al., 2019), suggesting that a dosing 
regimen involving 2 or 3 doses may mitigate the potential GI effects that were observed with consumption 
of 1 single bolus dose of (R, S)-1,3-butanediol.   
1.6 
Dose Rationale and Risk/Benefits 
 
The study dose was chosen based on the level of (R)-1,3-butanediol that was determined to be GRAS for 
use.  Thus, during the test day, study participants will consume 3 servings of Avela™; each serving will 
provide 11.5 g of (R)-1,3-butanediol.  Effects on ketone (BHB) production, GI tolerance, and 
sleepiness/alertness will be assessed.  
2 
Study Objectives 
2.1 
Primary Objective 
To evaluate the level of production of BHB following the consumption of 3 servings of Avela™, with 
consumption of each serving separated by 30 minutes and each serving providing 11.5 g of (R)-1,3-
butanediol [total intake of 34.5 g of (R)-1,3-butanediol].   
2.2 
Secondary Objectives (if applicable) 
To evaluate the acute effects of 3 servings of Avela™, with consumption of each serving separated by 30 
minutes and each serving providing 11.5 g of (R)-1,3-butanediol [i.e., total intake of 34.5 g (R)-1,3-
butanediol] on: 
o 
GI tolerance 
o 
Sleepiness/alertness 
3 
Study Design  
3.1 
General Design 
The clinical trial described herein is an open-label, acute study involving a total of 30 healthy male and 
female adults (ages 18 to 65 years; BMI of 18 to <35.0 kg/m2, inclusive).     
 
Prior to the clinical study, subjects will be screened according to the study inclusion and exclusion criteria, 
using an online survey (Appendix A).  Subjects meeting all the inclusion criteria and none of the 
exclusion criteria will be invited to participate in the study (Table 3.1-1).  These subjects will be sent study 
equipment and materials, including 1 consent form.  Subjects will be asked to read the consent form and 
e-mail either the Research Associate or the Principal Investigator with any specific questions. If there are 
no questions, or once the questions have been resolved, those that wish to participate will be asked to 
sign the consent form and send a picture of the signed page to the Research Associate of the study. 

Effects of (R)-1,3-butanediol on ketone production  
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Therefore, the subject will maintain a copy of the signed consent form, as per the U.S. Food and Drug 
Administration’s regulations.  
   
On the day of the clinical study, subjects will follow instructions for study methodology (Appendix B).  
Subjects, who will have fasted overnight for 12 hours, will self-assess and record GI symptomology [using 
the modified visual analogue scale (mVAS) GI symptoms tool], alertness [using the Stanford Sleepiness 
Scale (SSS)], and blood BHB level (using the Keto Mojo BHB monitor), at time 0 minutes, as baseline 
measures, using an online study portal (Appendix C).  Subjects will then consume 3 servings of Avela™, 
each providing 11.5 g of (R)-1,3-butanediol, with the first serving consumed at 0 minutes, the second 
serving at 30 minutes, and the third serving at 60 minutes.  After the collection of baseline measures (at 0 
minutes), GI symptomology (assessed using the mVAS GI symptoms tool), alertness (assessed using the 
SSS), and blood BHB levels (using the Keto Mojo BHB monitor) will be assessed, again, at 30, 60, 90, 
120, 180, 240, and 300 minutes.  At each time point, it is important that the order of collection be 
maintained [i.e., 1. GI symptomology; 2. Alertness/sleepiness; 3. BHB level; and 4. Avela™ (when 
Avela™ is to be consumed; i.e., at 0, 30, and 60 minutes)].  After the assessments at 240 minutes are 
complete, subjects will consume 1 ALOHA Organic Plant Based Protein Bar (Caramel Sea Salt).  During 
the study, subjects will be asked to record their blood BHB levels and questionnaire results using the 
online portal (Appendix C).  The results will be recorded electronically, using a de-identified survey on 
Survey Monkey, designed to protect subject anonymity [i.e., the survey will not be affiliated with a name 
or any personal information; rather, each participant with have a unique identification number, which will 
be used to access the survey and upload the results of the study).  
 
Table 3.1-1 
Study Groups Included in Clinical Study 
Number of Subjects 
Investigational Product; Dose 
30 subjects (males and females) 
(R)-1,3-butanediol; 3 doses of 11.5 g/dose 
3.2 
Study Endpoints 
3.2.1 Primary Study Endpoint 
• 
The measure of blood ketones levels (i.e., blood BHB levels) at 0 minutes (baseline) and at 30, 
60, 90, 120, 180, 240 and 300 minutes of study.  
3.2.2 Secondary Study Endpoints 
Using questionnaires, the following secondary outcomes will be determined: 
 
• 
GI symptomology; and 
• 
Sleepiness/alertness. 
3.3 
Primary Safety Endpoints  
• 
Tolerability: unsolicited adverse events (AEs) 
4 
Study Population and Duration of Participation  
4.1 
Duration of Study Participation  
The duration of subject participation is 2 days; one day for screening and one day for Avela™ (R)-1,3-
butanediol consumption.  
4.2 
Total Number of Subjects and Sites  
The proposed clinical study is being conducted by Genomatica.  Thirty Genomatica employees (males 
and females) are being recruited for the study.  There is no sample size calculation, as there is no control 
group and the study is a pilot study, with the main objective of understanding ketone (BHB) levels 
following consumption of (R)-1,3-butanediol. 30 participants are to be included, due to the inherent 

Effects of (R)-1,3-Butanediol on producing blood ketone levels 
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variability in the GI symptomology questionnaires. Of note, in other studies of food ingredients (e.g., 
inulin1, sorbitol2, erythritol + fructose3) where GI tolerance was an outcome of interest, sample sizes 
ranged from 26 to 37 participants 
 
Genomatica employees who express interest in participating in the study and who meet the eligibility 
criteria for study inclusion will complete the study remotely, within their own homes, using equipment 
provided by Genomatica (i.e., lancets, sharps container, alcohol swabs, Keto Mojo BHB monitor and 
ketone test strips (and glucose test strips, not to be used in the study), bandages, pregnancy test [if 
applicable], measuring tape, three 4 oz. (118 mL) bottles of sealed water premixed with Avela™ [each 
providing 11.5 g of (R)-1,3-butanediol], 1 shot glass with the Avela™ logo, an ALOHA Organic Plant 
Based Protein Bar (Caramel Sea Salt), 1 duffel bag, 1 consent form, and study instructions.  All study-
related data will be entered and transmitted by each study participant using an electronic platform, using 
surveys that will protect the identity of each participant.   
4.3 
Inclusion Criteria 
• 
Males and females, 18 to 65 years of age 
• 
BMI 18 to <35.0 kg/m2, inclusive 
• 
Weight is ≥ 110 lbs 
• 
Subject is judged to be in good health on the basis of medical history 
• 
Subject is willing to fast overnight, prior to study start 
• 
Subject is willing to avoid alcohol and intense physical activity the day prior to and on the day of 
testing 
• 
Subject is capable of giving informed consent and complying with all study procedures/ 
requirements. 
4.4 
Exclusion Criteria 
• 
Previous GI disorders (e.g., inflammatory bowel disease, irritable bowel syndrome, history of 
surgery for weight loss, or gastroparesis 
• 
Gastroenteritis in the 2 weeks preceding the study 
• 
Weight <110 lbs 
• 
Diabetes 
• 
History of alcohol or drug abuse 
• 
Previous diagnosis of neurological disorders, depression, or mental illness with psychosis 
• 
Unexplained alarm features (i.e., unintentional weight loss >10% body weight in the last 3 
months, fevers, or blood in stools) 
• 
Use of an antibiotic or any medication impacting gut transit during the 2 weeks before the study 
• 
Constipation or diarrhea (defined as, on average, less than 3 stools per week or more than 3 
stools per day, respectively) 
• 
Allergy to tree nuts 
• 
Women who are pregnant or breastfeeding;  
• 
Persons with medical conditions affecting the pancreas, liver, thyroid, or gall bladder. 
4.5 
Subject Recruitment  
All subjects will be employees of Genomatica Inc., located in San Diego, California.  Participation in the 
study is not a requirement of employment, and measures are being taken to ensure that Genomatica 
employees do not feel coerced to partake in the study.  An employee’s decision about research 
participation will not affect (favorably or unfavorably) performance evaluations, career advancement, or 
other employment-related decisions made by peers or supervisors.       
 
1 Example study (Bonnema et al., 2010) available at: https://doi.org/10.1016/j.jada.2010.03.025  
2 Example study (Fernández-Bañares et al., 2005) available at:  https://pubmed.ncbi.nlm.nih.gov/16410032/ 
3 Example study (Kim et al., 2011) available at: https://pubmed.ncbi.nlm.nih.gov/22118754/  

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4.6 
Early Withdrawal of Subjects  
4.6.1 When and How to Withdraw Subjects 
Subjects may withdraw consent or be removed from the study by the Principal Investigator (PI).  In the 
event that a subject is withdrawn from the study, the reason for the withdrawal will be documented, with 
the primary reason selected from the following standard categories: 
 
• 
Withdrawal of consent: the subject requests to withdraw from further participation in the study in 
the absence of a medical need to withdraw as determined by the PI. 
• 
Lost to follow-up: for unknown reasons, the subject did not upload their study results on to the 
electronic platform. 
• 
Poor compliance: the subject consumes only 1 or 2 of the 3 servings of the study product; is 
found to meet an exclusionary criterion; and/or is not complying with other study obligations. 
• 
AE: in the judgment of the PI and in the best interests of the subject, events that require 
discontinuation of study product (includes all categories of study product relatedness: None, 
Remote, Possible, Probable, or Highly Probable). 
• 
Death: death of the subject. 
• 
Other: causes of premature termination from the study other than the above, such as theft or loss 
of study products, termination of study by Sponsor, etc. 
4.6.2 Data Collection and Follow-up for Withdrawn Subjects 
Should a subject request to withdraw or be withdrawn from the study by the PI, the subject’s reason for 
withdrawal will be documented.   
5 
Study Product  
5.1 
Description and Treatment Regimen 
An outline of the study timepoints is provided in Table 5.1-1.  At Screening, subject eligibility will be 
determined using an online survey (Appendix A).  Participants will be enrolled in the study in groups of 
10 at a time.  Participants will eat a meal the night before the study and begin the study the next morning 
in a fasted state (i.e., overnight, 12-hr fast).  The time at which the last meal was consumed and the study 
start time should be recorded using the online portal (Appendix C).     
 
The study will be approximately 300 minutes (5 hours), from start to finish.  Completion of study 
questionnaires, assessment of blood BHB levels, and consumption of the Avela™ beverage will be 
completed, as per Table 5.1-1; a detailed description of the methodology for each study timepoint is 
provided in section 6.0 of this clinical study protocol.  
 

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Table 5.1-1 
Clinical Study Outline  
Assessment 
Tool Utilized 
Time (min) 
Screening 
(up to 7 
days prior 
to test day) 
0 
30 
60 
90  
120 
180 
240 
300 
Eligibility for 
study 
participation  
On-line Survey 
X 
- 
- 
- 
- 
- 
- 
- 
- 
GI 
symptomatology 
mVAS GI 
Symptoms 
Tool 
- 
X 
X 
X 
X 
X 
X 
X 
X 
Sleepiness/ 
Alertness 
Assessment 
Stanford 
Sleepiness 
Scale 
- 
X 
X 
X 
X 
X 
X 
X 
X 
Blood BHB levels 
Keto-Mojo 
Monitor 
- 
X 
X 
X 
X 
X 
X 
X 
X 
(R)-1,3-
butanediol 
consumption 
Avela™ 
- 
X 
X 
X 
- 
- 
- 
- 
- 
ALOHA Bar 
consumption 
Not applicable 
 
- 
- 
- 
- 
- 
- 
X 
- 
BHB = beta-hydroxybutyrate; GI = gastrointestinal; min = minutes; mVAS = modified visual analogue scale. 
5.2 
Method for Assigning Subjects to Treatment Groups  
All subjects included in the study will be assigned to receive the active investigational product [Avela™ 
(R)-1,3-butanediol] (i.e., there is only one group for the study, with a before, after study design).  Upon 
enrolment, each participant will be provided with a unique identification number. 
5.3 
Subject Compliance Monitoring  
Compliance of subjects will be assessed by using compliance records.  Participants must consume all 3 
doses of product, at the specified timepoints, to meet compliance requirements. 
5.4 
Prior and Concomitant Therapy  
Subjects may continue their use of medications and/or dietary supplements throughout the study period, 
so long as they do not have conditions or are taking medications are not contra-indicated (see Exclusion 
Criteria in Section 4.4).   
5.5 
Handling of Study Product  
5.5.1 Dispensing of Study Product  
At screening, all subjects included in the study will be provided with the following: 
 
• 3 x 4 oz (118 mL) bottles of water premixed with Avela™ [each providing 11.5 g of (R)-1,3-
butanediol]; 
• Lancets; 
• Alcohol swabs; 
• 1 sharps container; 
• 1 shot glass with the Avela™ logo; 
• 1 Keto Mojo BHB monitor and ketone test strip; 
• Bandages; 
• 1 pregnancy test [if applicable]; 

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• 1 x measuring tape; 
• 1 x ALOHA Organic Plant Based Protein Bar (Caramel Sea Salt); 
• 1 duffel bag; 
• 1 x study consent form; and 
• 1 x study instruction sheet (Appendix B). 
5.5.2 Return or Destruction of Study Product  
The PI will maintain records of the product's delivery to the study participants and its use by each subject.  
These records should include dates, quantities, batch/serial numbers, expiration dates (if applicable), and 
the unique code numbers assigned to the trial subjects.  Investigators should maintain records that 
document adequately the doses provided to subjects and consumed by subjects so as to permit complete 
reconciliation of all investigational product(s).  As the study product is a lawful food ingredient in the 
United States (U.S.), there will be no requirement for study participants to return unused study product to 
Genomatica.  Rather, any unused study product can be disposed of by the participant by flushing down 
the toilet or pouring down the drain. The sharps container is provided with a prepaid outer shipping carton 
and water-resistant sealing tape and a shipping manifest form for tracking and certificate of destruction. 
Study subjects will follow the mailing instructions provided with the sharps container to return the 
container. 
6 
Study Schedule 
A tabular summary of the schedule of procedures is provided in Table 5.1-1.  An overall review of the 
order of events on the test day is provided in Table 6-1.  A breakdown of the order of events at each 
study timepoint (i.e., prior to study start and at 0, 30, 60, 90, 120, 180, 240, and 300 minutes) is provided 
in Sections 6.2.1 to 6.3.0. 
 

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Table 6-1 
Order of Events on Study Test Day  
Timepoint 
(min) 
mVAS GI 
Symptoms Tool 
SSS  
 
BHB Measure  
Consumption of 
Avela™ 
Consumption 
of ALOHA Bar 
0 
1 
2 
3 
4 
- 
30 
1 
2 
3 
4 
- 
60 
1 
2 
3 
4 
- 
90 
1 
2 
3 
- 
- 
120 
1 
2 
3 
- 
- 
180 
1 
2 
3 
- 
- 
240 
1 
2 
3 
- 
4 
300 
1 
2 
3 
- 
- 
 
6.1 
Screening – Day 1 (Investigative Staff) 
• 
Assess participant eligibility using responses to online survey (Appendix A). 
• 
If participant is eligible, obtain signature of subject on study Informed Consent Form for study 
participation. 
• 
Provide participants with three 4 oz (118 mL) bottles of water premixed with Avela™ [each 
providing 11.5 g of (R)-1,3-butanediol]; lancets; alcohol swabs; 1 Keto Mojo BHB monitor; 
bandages; measuring tape; pregnancy test [if applicable]; one ALOHA Bar; 1 x study consent 
form; and 1 x study instruction sheet (Appendix B). 
6.2 
Study Test Day – Day 2 (Study Participant) 
6.2.1 Prior to Study Start 
• 
Pregnancy test [if applicable] 
o 
If a participant answered “no” to both of the study screening questions: 
§ 
“If you are a female, are you post-menopausal, defined as not having had a 
period for 12 consecutive months or longer?” 
§ 
“If you are a female, have you had any procedure that would result in you not 
being able to have children, such as “tying of tubes” or a removal of your 
ovaries?” 
o 
Then the subject is to take the pregnancy test prior to study start and proceed only if the 
test is negative (the result of the test will be included on the online portal). 
 
• 
Setting up the Keto-Mojo Monitor 
o 
The subject will be instructed to download the MyMojoHealth app from the Apple Store or 
Google Play.  
o 
The subject will sync the app with his/her Keto-Mojo Monitor. 
o 
The study coordinator will send the subject a request, on his/her App, to join the study 
account, in order to share all BHB readings acquired during the study.  Each subject’s 
data will be downloaded via the account. 
• 
The subject will familiarize themselves with the online system for data recording. 
6.2.2 0 min timepoint 
• 
Using the online portal, the subject will: 
o 
Record the time their last meal was consumed.  
o 
Record the start time of the study. 
o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 

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• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal. 
• 
Start their on-screen timer. 
• 
Consume one (1) bottle of premixed Avela™ liquid within a 5-minute time period.   
6.2.3 30 min timepoint 
• 
Using the online portal, the subject will: 
o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 
• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal. 
• 
Consume one (1) bottle of premixed Avela™ liquid within a 5-minute time period.  
6.2.4 60 min timepoint 
• 
Using the online portal, the subject will: 
o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 
• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal.  
• 
Consume one (1) bottle of premixed Avela™ liquid within a 5-minute time period.   
6.2.5 90 min timepoint 
• 
Using the online portal, the subject will: 
o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 
• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal. 
6.2.6 120 min timepoint 
• 
Using the online portal, the subject will: 
o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 
• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal. 
6.2.7 180 min timepoint 
• 
Using the online portal, the subject will: 
o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 
• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal. 
6.2.8 240 min timepoint 
• 
Using the online portal, the subject will: 
o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 
• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal. 
• 
Consume the provided ALOHA Bar within 7 minutes.   
6.2.9 300 min timepoint 
• 
Using the online portal, the subject will: 

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o 
Complete the mVAS GI symptoms tool. 
o 
Complete the SSS. 
• 
Measure their blood BHB level using the Keto Mojo Monitor and record the value on the online 
portal. 
6.3.0 End of Study  
Subjects will be instructed to connect to the Keto Mojo App to allow it to sync with the Keto-Mojo Monitor. 
Once the study is complete, the subject will be instructed to close all browser windows and to dispose of 
any unused study product (either by flushing down the toilet or pouring down the drain). The sharps 
container can be mailed using the provided return envelope for disposal.   
7 
Study Procedures 
7.1 
Clinical Evaluations 
There are no clinical evaluations to be completed as part of this study protocol.  
7.2 
Laboratory Evaluations 
7.2.1 Clinical Laboratory Evaluations: Blood BHB Measurement 
To determine blood BHB levels, each participant will be provided with a Keto-Mojo Monitor4 (Keto-Mojo, 
Amsterdam, Netherlands).  The monitor allows for each subject to measure their own ketone levels (i.e., 
BHB levels), through sampling of capillary blood.  Moore et al. (2021) tested the Keto-Mojo meter for 
accuracy versus the Abbott Lab Precision Xtra glucose/ketone device (Abbott’s ketone meter) and 
reported there was excellent agreement between the two meters.  Abbott’s ketone meter has been 
validated in comparison to mass spectrometry (the gold standard of ketone measurement) in animal 
studies (Pineda and Cardoso, 2015; Bach et al., 2016) and in humans at ketone levels below 3 mM 
(Janssen et al., 2010; Yu et al., 2011).  In their publication, Moore et al. (2021) indicate:  
 
“For the purpose of identifying nutritional ketosis, the Keto-Mojo device (Meter 2) is a 
reliable and accurate portable blood ketone measurement device that is more cost-
effective than another commonly used portable ketone meter, the Precision Xtra (Meter 
1). The acceptable measurement error, consistent level of agreement, and high 
diagnostic performance of Meter 2 compared to Meter 1 makes it appropriate for use by 
recreational or professional athletes interested in verifying that they are in a state of 
[nutritional ketosis] on a daily basis.” 
 
Capillary blood BHB levels will be measured at baseline (0 min) and at 30, 60, 90, 120, 180, 240, and 300 
minutes of the study.  A total of 10 fingersticks will be taken [8 study timepoints + 2 control timepoints 
(maximum)]. A typical blood drop contains ~35 µl of blood5.  It is likely that 2 to 3 drops, per fingerstick, 
will be required (i.e., 105 µl of blood). Total blood collection during the study will be: (8+2) X 105 µl = 
1,050 µl or 1.050 ml. Frequent fingerstick blood sampling is common when examining post-prandial 
glycaemic responses, as capillary blood is more ideal (subject to less variability) than venous blood, as 
per the International Organization for Standardization (ISO 26642:20106). Also, as per this standard, the 
number of fingerstick blood sampling is 8, which is very similar to the number in our protocol. Although 
our interest is in post-prandial ketone (BHB) responses, the analogy is relevant with respect to the 
acceptability of the blood sampling schedule and frequency of blood draws. 
 
4 Available at: https://keto-mojo.com/video/why-keto-mojo-is-the-gold-standard-for-testing-blood-ketones/.  
5 Available at: Not Every Drop of Blood Is Identical | The Partnership for Global Health Technologies (bu.edu)  
6 Available at: https://www.iso.org/standard/43633.html 

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7.3 
Questionnaire Assessments 
7.3.1 GI Symptoms Assessment 
A validated tool to measure GI tolerability of (R)-1,3-butanediol will be utilized.  The mVAS GI symptoms 
tool is a self-administered questionnaire, which is modelled after the validated “gold standard” 
measurement tool predominantly used to test clinical gastroenterology scenarios (Bengtsson et al., 2013).  
Gaskell et al. (2019) tested the reliability of the mVAS in assessing GI symptoms during exercise, with 
and without dietary interventions.  The mVAS has been modified slightly for this clinical study protocol, as 
there is no exercise employed in this study.  The mVAS uses a 10-point rating scale to self-report on GI 
symptoms; 1–4 is indicative of mild GI symptoms (i.e., sensation of GI symptoms, but not substantial 
enough to interfere normal activities); 5–9 is indicative of severe GI symptoms (i.e., GI symptoms 
substantial enough to interfere with normal activities), and 10 is indicative of extremely severe GI 
symptoms warranting cessation of all other activities.  If no specific GI symptom is reported, this would be 
recorded as a 0.  A sample of the questionnaire is available within the screen captures of the online portal 
(Appendix C).  In the study by Gaskell et al. (2019), GI symptoms, such as regurgitation and defecation, 
were rated dichotomously, either as 0 (no symptom) if they did not result in a cessation of exercise or as 
10 (extremely severe symptoms) if they resulted in either a temporary or complete cessation of exercise; 
in this clinical study, these have been modified to a full 10-point VAS scale.  All subjects will complete the 
mVAS at baseline (0 minutes), and at 30, 60, 90, 120, 180, 240, and 300 minutes. 
7.3.2 Assessment of Alertness 
The SSS (Shahid et al., 2012) is a subjective tool to assess how alert a subject is feeling at specific 
moments in time.  The scale requires respondents to select a rating of 1 to 7, where a “1” indicates the 
subject is “feeling active, vital, alert, or wide awake” and the highest score of “7” indicates the subject is 
“no longer fighting sleep, sleep onset soon; having dream-like thoughts” (Hoddes et al., 1973).  The scale 
is validated, correlating r = 0.68 with performance on the Wilkinson tests.  All subjects will complete the 
SSS at baseline (0 minutes) and at 30, 60, 90, 120, 180, 240, and 300 minutes. The SSS is available in 
the screen captures of the online portal (Appendix C). 
8 
Statistical Analysis Plan 
Statistical analyses to be conducted will be completed by the data management team. 
9 
Safety and Adverse Events 
9.1 
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others 
Any incident, experience, or outcome that meets all of the following criteria:  
• 
Unexpected in nature, severity, or frequency [i.e., not described in study-related documents such 
as the Institutional Review Board (IRB)-approved protocol or consent form]; 
• 
Related or possibly related to participation in the research (i.e., 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 greater risk of harm (including physical, 
psychological, economic, or social harm). 
 
Serious Adverse Event 
AEs are classified as serious or non-serious.  A serious adverse event (SAE) is any AE that is:  
 
• 
fatal 
• 
life-threatening 
• 
requires or prolongs hospital stay 
• 
results in persistent or significant disability or incapacity 
• 
a congenital anomaly or birth defect 

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• 
an important medical event 
 
Important medical events are those that may not be immediately life-threatening, but are clearly of major 
clinical significance.  They may jeopardize the subject, and may require intervention to prevent one of the 
other serious outcomes noted above.  For example, a seizure that did not result in in-patient 
hospitalization, or intensive treatment of bronchospasm in an emergency department would typically be 
considered serious.  
 
All AEs that do not meet any of the criteria for serious should be regarded as non-serious AEs.  
 
Adverse Event Reporting Period 
The study period during which AEs must be reported is normally defined as the period from the initiation 
of any study procedures to the end of the study treatment follow-up.  For this study, the study treatment 
follow-up is defined as the evening of the day on which the clinical study takes place.  
 
Pre-existing Condition 
A pre-existing condition is one that is present at the start of the study.  A pre-existing condition should be 
recorded as an AE if the frequency, intensity, or the character of the condition worsens during the study 
period. 
 
General Physical Examination Findings 
As the study product is a food ingredient that is GRAS in the U.S., there are no general examinations 
planned for this clinical study.  
 
Post-study Adverse Event 
All unresolved AEs should be followed by the investigator until the events are resolved, the subject is lost 
to follow-up, or the AE is otherwise explained.  While adverse events related to GI intolerance are being 
solicited, the study participants will also be able to provide information on any other adverse events 
experienced at the end of the mVAS questionnaire, where space is provided for detailing “other” adverse 
events, including those that are not GI related.   
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any AE that results in hospitalization or prolonged hospitalization should be documented and reported as 
a SAE unless specifically instructed otherwise in this protocol.  Any condition responsible for surgery 
should be documented as an AE if the condition meets the criteria for an AE.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an AE if 
hospitalization or prolonged hospitalization is for diagnostic or elective surgical procedures for a 
preexisting condition.  Surgery should not be reported as an outcome of an AE if the purpose of the 
surgery was elective or diagnostic and the outcome was uneventful. 
9.2 
Recording of Adverse Events 
Although AEs are not expected, should any non-serious or serious AEs occur, it is recommended that the 
participant seek immediate treatment.  All AEs occurring during the study period must be recorded.  The 
clinical course of each event should be followed until resolution, stabilization, or until it has been 
determined that the study treatment or participation is not the cause.  SAEs that are still ongoing at the 
end of the study period must be followed up to determine the final outcome.  Any SAE that occurs after 
the study period and is considered to be possibly related to the study treatment or study participation 
should be recorded and reported to the IRB immediately. 
9.3 
Reporting of Serious AEs and Unanticipated Problems 
The Investigator must conform to the AE reporting timelines, formats and requirements of the various 
entities to which they are responsible, but at a minimum those events that must be reported are those that 
are: 
 

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• 
related to study participation, 
• 
unexpected, and  
• 
serious or involve risks to subjects or others  
• 
(see definitions, section 9.1).   
 
If the report is supplied as a narrative, the minimum necessary information to be provided at the time of 
the initial report includes: 
 
• 
Subject number 
• 
A description of the event 
• 
Date of onset 
• 
Current status 
• 
Whether study treatment was discontinued 
• 
The reason why the event is classified as serious 
• 
An assessment of the association between the event and study treatment by a qualified person, 
as needed 
9.3.1 Investigator reporting: Notifying the IRB 
Investigators are responsible for safety reporting to their local IRB.  Investigators are responsible for 
complying with their local IRB’s reporting requirements, though must submit the required reports to their 
IRB no later than 10 working days.  Copies of each report and documentation of IRB notification and 
receipt will be kept in the investigator’s study file. 
10 Study Administration, Data Handling and Record Keeping 
10.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the requirements of 
the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a 
signed subject authorization informing the subject of the following:  
 
• 
What protected health information (PHI) will be collected from subjects in this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of their PHI 
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, 
retains the ability to use all information collected prior to the revocation of subject authorization.   
10.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other activities in a 
clinical trial necessary for the reconstruction and evaluation of the trial.  Source data are contained in 
source documents.  In the case of the clinical trial proposed herein, examples of original documents and 
data records include: consent forms and the online study portal data (blood BHB levels and 
questionnaires). 
10.3 Records Retention 
Genomatica will retain records for this clinical trial for a period of 2 years from completion of the trial, 
defined as the day the final subject completes the study.  The files will be stored with Human 
Resource/Legal/Finance files – locked with restricted access; at 4757 Nexus Centre Drive, San Diego CA 
92121. 

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11 Study Monitoring, Auditing, and Inspecting 
11.1 Study Monitoring Plans 
The clinical study described herein is a pilot study intended to determine the level of ketosis (BHB) 
achievable with consumption of the food ingredient, (R)-1,3-Butanediol.  The study is not being conducted 
in accordance with Good Clinical Practice, and therefore, there is no clinical study monitoring. 
11.2 Auditing and Inspecting 
The clinical study described herein is a pilot study intended to determine the level of ketosis (BHB) 
achievable with consumption of the food ingredient, (R)-1,3-Butanediol.  The study is not being conducted 
in accordance with Good Clinical Practice, is not a regulated pharmaceutical trial, and therefore, will not 
be audited or inspected. 
12 Ethical Considerations 
This protocol and any amendments will be submitted to a properly constituted independent Ethics 
Committee (EC) or IRB, in agreement with local legal prescriptions, for formal approval of the study 
conduct.  The decision of the EC/IRB concerning the conduct of the study will be made in writing. 
 
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study.  This consent 
form will be submitted with the protocol for review and approval by the EC/IRB for the study.  The formal 
consent of a subject, using the EC/IRB-approved consent form, must be obtained before that subject 
undergoes any study procedure.  The consent form must be signed by the subject or legally acceptable 
surrogate.  
 
The subjects for the study proposed herein are being recruited from the pool of employees at 
Genomatica.  Participation in the study is not a requirement of employment, and measures are being 
taken to ensure that Genomatica employees do not feel coerced to partake in the study.  For example, 
employees will not be directly solicited to enroll in research, neither orally nor through individual mailings 
or individual e-mails.  Acceptable methods of recruitment will include flyers and other recruiting materials 
(approved by an IRB), displayed in the workplace where public announcements are permitted and/or 
mass e-mailers.  The IRB will be provided with specific plans for ensuring the privacy of employees that 
participate in research.  Also, policies will be clearly spelled out in the informed consent for issues such 
as injury or sickness as a result of the research.  It will be made clear to employees that an employee’s 
decision about research participation will not affect (favorably or unfavorably) performance evaluations, 
career advancement, or other employment-related decisions made by peers or supervisors.         
13 Study Finances 
13.1 Funding Source 
This study will be financed by Genomatica Inc.  
13.2 Subject Stipends or Payments 
All participants will be provided with a Keto-Mojo ketone/glucose monitor kit (complete with ketone and 
glucose test strips, lancets, alcohol swabs, sharps container and bandages), a measuring tape, a shot 
glass with the Avela™ logo and a duffel bag, which they are permitted to keep, following the study.  There 
will no costs incurred by subjects.   
14 Publication Plan 
Genomatica Inc. plans to register this study at ClinicalTrials.gov and to publish the results of the study. 

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15 References 
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ketosis and may improve cognition in Alzheimer's disease. A systematic review and meta-
analysis of human studies. Ageing Research Reviews, 58, 101001 [11pp]. 
DOI:10.1016/j.arr.2019.101001. 
 
Bach KD, Heuwieser W, McArt JAA (2016). Technical note: Comparison of 4 electronic handheld meters 
for diagnosing hyperketonemia in dairy cows. J Dairy Sci 99(11):9136-9142. 
DOI:10.3168/jds.2016-11077. 
Bengtsson, M., Persson, J., Sjölund, K., & Ohlsson, B. (2013). Further validation of the visual analogue 
scale for irritable bowel syndrome after use in clinical practice. Gastroenterology Nursing, 36, 
188–198. PubMed ID: 23732784 doi:10.1097/SGA.0b013e3182945881 
Cahill GF Jr (1970). Starvation in man. New Engl J Med 282(12):668-675. 
DOI:10.1056/NEJM197003192821209. Cited In: Veech et al., 2001. 
Desrochers S, David F, Garneau M, Jetté M, Brunengraber H (1992). Metabolism of R-and S-1,3-
butanediol in perfused livers from meal-fed and starved rats. Biochem J 285(2, Part 2):647-653. 
DOI:10.1042/bj2850647. 
Gaskell, S, Snipe R, Costa R. (2019). Test–Retest Reliability of a Modified Visual Analog Scale 
Assessment Tool for Determining Incidence and Severity of Gastrointestinal Symptoms in 
Response to Exercise Stress. Int J Sport Nutr and Exer Metab 29:411-419. 
Grove R, Prapavessis H (1992). Preliminary evidence for the reliability and validity of an abbreviated 
Profile of Mood States. Int J Sport Psychol 23:93-109. 
Hoddes E, Zarcone V, Smythe H, Phillips R, Dement WC (1973). Quantification of sleepiness: a new 
approach. Psychophysiology 10(4):431-436. DOI:10.1111/j.1469-8986.1973.tb00801.x. 
Janssen MJ, Hendrickx BH, Habets-van der Poel CD, van den Bergh JP, Haagen AA, Bakker JA (2010). 
Accuracy of the Precision® point-of-care ketone test examined by liquid chromatography tandem-
mass spectrometry (LC-MS/MS) in the same fingerstick sample. Clin Chem Lab Med 
48(12):1781-1784. DOI:10.1515/CCLM.2010.351. 
Kies C, Tobin RB, Fox HM, Mehlman MA (1973). Utilization of 1,3-butanediol and nonspecific nitrogen in 
human adults. J Nutr 103(8):1155-1163. DOI:10.1093/jn/103.8.1155. 
McNair D, Lorr M, Droppleman L (1971). Manual for the Profile of Mood States. San Diego (CA): 
Educational and Industrial Testing Service (EdITS). Cited In: Grove and Prapavessis, 1992. 
McNair D, Lorr M, Droppleman L (1992). Profile of Mood States Manual, revised edition. San Diego (CA): 
Educational and Industrial Testing Service (EdITS). Cited In: van Wijk, 2011. 
Moore AR, Holland-Winkler AM, Ansley JK, Boone EDH, Schulte MKO (2021). Reliability and diagnostic 
performance of a new blood ketone and glucose meter in humans. J Int Soc Sports Nutr 18(1):6 
[9pp]. DOI:10.1186/s12970-020-00404-2. 
Pineda A, Cardoso FC (2015). Technical note: Validation of a handheld meter for measuring β-
hydroxybutyrate concentrations in plasma and serum from dairy cows. J Dairy Sci 98(12):8818-
8824. DOI:10.3168/jds.2015-9667. 

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Poff AM, Rho JM, D'Agostino DP (2019). Ketone administration for seizure disorders: history and 
rationale for ketone esters and metabolic alternatives. Front Neurosci 13:1041 [13pp]. 
DOI:10.3389/fnins.2019.01041. 
Reichard GA Jr, Owen OE, Haff AC, Paul P, Bortz WM (1974). Ketone-body production and oxidation in 
fasting obese humans. J Clin Invest 53(2):508-515. DOI:10.1172/JCI107584. 
Scott BE, Laursen PB, James LJ, Boxer B, Chandler Z, Lam E, et al. (2019). The effect of 1,3-butanediol 
and carbohydrate supplementation on running performance. J Sci Med Sport 22(6):702-706. 
DOI:10.1016/j.jsams.2018.11.027. 
Shahid A, Wilkinson K, Marcu S, Shapiro CM, editors (2012). Stanford Sleepiness Scale (SSS). In: 
STOP, THAT and One Hundred Other Sleep Scales. New York (NY): Springer Science+Business 
Media, LLC, pp. 369-370. 
Shaw DM, Merien F, Braakhuis A, Plews D, Laursen P, Dulson DK (2019). The effect of 1,3-butanediol on 
cycling time-trial performance. Int J Sport Nutr Exerc Metab 29(5):466-473. 
DOI:10.1123/ijsnem.2018-0284. 
Shivva V, Cox PJ, Clarke K, Veech RL, Tucker IG, Duffull SB (2016). The population pharmacokinetics of 
D-β-hydroxybutyrate following administration of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate. AAPS 
J 18(3):678-688. DOI:10.1208/s12248-016-9879-0. 
Tobin RB, Mehlman MA, Kies C, Fox HM, Soeldner JS (1975). Nutritional and metabolic studies in 
humans with 1,3-butanediol. Fed Proc 34(12):2171-2176. 
Van Wijk C (2011). Mood States are not associated with BMI in mentally healthy adults. Psychology 
2(5):492-496. DOI:10.4236/psych.2011.25076. 
Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill GF Jr (2001). Ketone bodies, potential therapeutic 
uses. IUBMB Life 51(4):241-247. DOI:10.1080/152165401753311780. 
Yu H-YE, Agus M, Kellogg MD (2011). Clinical utility of Abbott Precision Xceed Pro® ketone meter in 
diabetic patients. Pediatr Diabetes 12(7):649-655. DOI:10.1111/j.1399-5448.2011.00768.x 
16 Appendices 
Appendix A 
Screen Captures of Online Screening Survey  
Appendix B 
Detailed Instructions for Participants 
Appendix C 
Screen Captures of Online Portal  

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Appendix A Screen Captures of Online Screening Survey  

Effects of (R)-1,3-Butanediol on producing blood ketone levels 
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Appendix B Detailed Instructions for Participants  
B-1 Detailed instructions for participants 
 
Day prior to study start 
1. Final meal should be consumed to ensure a minimum of a 12-hour fast prior to study start.  You 
are permitted to drink water when you wake up; do not consume any food.  Record the time at 
which the last meal was consumed using the online portal. 
2. Place Avela™ test beverages in the fridge for consumption of chilled beverages on study date. 
3. Download the Keto-Mojo App from the Apple Store or Google Play and make sure it is paired with 
your Keto-Mojo Monitor. 
4. You will be sent a request on your App to join the study account, in order to share all BHB 
readings acquired during the study; please accept.  
5. Test blood BHB levels  
o 
Use the Keto-Mojo monitor to measure your blood BHB level.  The manual provided with 
the monitor provides step by step instructions. Alternatively, you can review these videos 
for assistance:  
• 
How to take a reading on the Keto-Mojo meter: https://keto-mojo.com/video/gkplus-
meter-testing-demo/ 
• 
How to use the lancet device: https://keto-mojo.com/video/gkplus-meter-lancet-
device/ 
 
Study Start 
6. Please ensure 12 hours have elapsed since the last food product was consumed. 
7. Retrieve from the fridge the bottles of water containing the premixed Avela™ product. 
8. On the day of study, if you are a female and you answered “no” to both of these questions during 
the study screening: 
i. “If you are a female, are you post-menopausal, defined as not having had a 
period for 12 consecutive months or longer?” and 
ii. “If you are a female, have you had any procedure that would result in you not 
being able to have children, such as “tying of tubes” or a removal of your 
ovaries?” 
Take the pregnancy test provided prior to study start. Proceed with the study only if the test is 
negative (the result of the test will be included on the online portal). 
 
Baseline (0 minutes) 
9. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.   
10. The on-screen timer will start automatically upon reading the welcome screen and completing 
age, height, last meal questions. 
11. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal.  
12. Consume one of the Avela™ beverages within 5 minutes.   
 
30 minutes 
13. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.   
14. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal. 
15. Consume one of the Avela™ beverages within 5 minutes.   
 
60 minutes 
16. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.    
17. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal.  
18. Consume one of the Avela™ beverages within 5 minutes.   
 

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90 minutes 
19. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.   
20. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal. 
 
120 minutes 
21. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.   
22. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal. 
 
180 minutes 
23. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.   
24. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal. 
 
240 minutes 
25. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.   
26. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal. 
27. Consume the ALOHA Bar that has been provided, within a 7-minute timeframe. 
 
300 minutes 
28. Using the online portal, complete the mVAS for GI symptoms, followed by the SSS.   
29. Use the Keto-Mojo monitor to measure your blood BHB level and record this value on the online 
portal. 
 
Congratulations!  You have now completed the study!  Please open the Keto-Mojo App to allow it to sync 
with the Keto-Mojo Monitor and mail back the sharps container using the prepaid outer shipping carton, 
when convenient.  
. 

 
 
Appendix C Screen Captures of Online Portal