GRAS Notice GRN 969 Bacillus Subtilis

GRAS Notice (GRN) No.
969
https://www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory
.Ai. AlBMR Life Sciences, Inc.
July 21, 2020
Susan Carlson, PhD SEP 1 6 '. 320

Division Director
Division of Biotechnology and GRAS Notice Review OFFICE OF FOOD ADDITIVE SAFETY
Office of Food Additive Safety (HFS-200)
Center for Food Safety and Applied Nutrition
Food and Drug Administration
Department of Health and Human Services
5001 Campus Drive
College Park, MD 20740
Dear Dr. Carlson:

In accordance with regulation 21 CFR Pa1i 170 Subpart E (Generally Recognized
as Safe (GRAS) Notice), on behalf of Danisco USA, Inc. (the notifier), the
undersigned, Jessica Gruber, submits, for FDA review, the enclosed notice that
Bacillus subtilis Bss-19 is GRAS under the conditions of its intended use in foods.
Should you have any questions or concerns regarding this notice, please contact me
at 253-286-2888 or jessica@aibmr.com.
Sincerely,
Jessica Gruber, ND (agent of the notifier)

Scientific and Regulatory Consultant
AIBMR Life Sciences, Inc. ("AIBMR")
Bacillus subti/is Bss-19 GRAS 2

September 11, 2020
Dr. Carlson and staff,
To prevent any potential confusion, this submission replaces one that should have
arrived at your office on July 29, 2020, but apparently was lost by FedEx.
If you did indeed receive that first shipment, please disregard this duplicate.
Thank you!
Jared
Jared Douglas Brodin

Director of Information Services
AIBMR Life Sciences, Inc.
Notice to US Food and Drug Administration of the
Conclusion that the Intended Use of Bss-19 is
Generally Recognized as Safe
Submitted by the Notifier:

Danisco USA, Inc.
DuPont Nutrition & Biosciences
Four New Century Parkway
New Century, Kansas 66031
Prepared by the Agent of the Notifier:

AIBMR Life Sciences, Inc
1425 Broadway, Suite 458
Seattle WA 98122
July 21, 2020

A AIBMR Life Sciences, Inc.
Table of Contents
Part 1: Signed Statements and Certification ............................................................ 6

1. I Submission of GRAS Notice .............................. ............. ......... ... .................. 6
1.2 Name and Address of the Notifier and Agent of the Notifier ................. ... .... 6
1.3 Name of the Substance .......................................... ... ... ................................... 6
1.4 Intended Conditions of Use ........ ..... .. ..... ... ......... ........ ............. ... .................... 6
1.5 Statutory Basis for GRAS Conclusion .... .................... .... ..................... ..... ..... 7
1.6 Not Subject to Premarket approval ... .................... ......................................... 7
1.7 Data and Information Availability Statement ................................................ 7
1.8 Exemption from Disclosure under the Freedom oflnformation Act.. ........... 7
1. 9 Certification of Completion ........................ ................................................... 8
Part 2: Identity, Method of Manufacture, Specifications, and Physical or Technical
Effect ............. ... ........ ..... ....... ... ..... ... .. .................................................. ..................... 9
2.1 Identification ......... .. ... ...... .. .. ..... .. ................... .. .............. ......... ... ............ ........ 9
2.1.1 Taxonomy of Bss-19 ................... .. .......................................................... 9
2.1.2 Genetic Identification of Bss-19 ........ ... ............... .. ............................. ..... 9
2.2 Manufacturing ........................................... .. ............... .................................. 10
2.2.1 Good Manufacturing Practice ................................................................ 10
2.2.2 Raw Materials ........................................................................................ 10
2.2.3 Manufacturing Narrative and Flowchart ............ ...... ....... .. .................... 10
2.3 Specifications ................................ ..................................... ................ .......... 14
2.3.1 BatchAnalysis ... ......................................................... .................... .. ..... 15
2.3.2 Residual Pesticide Analysis .... ....... ........... ............................. ........... ..... 16
2.3.3 Shelf- Life Stability ............................................................................... 16
2.4 Antibiotic Resistance .......... ...... ......... .... ............................................. ..... .... 16
2.5 Genomic Analysis for Virulence and Pathogenicity .................................... 18
2.6 Resistance to Gastric Acidity and Bile Salts ................................................ 18
2.7 Hemolysis ............... ...................................................................................... 19
2.8 Biogenic Amine Formation ......................................................... .. ............... 20
2.9 D(-)/L(+)-Lactic Acid Production ......................................... ....................... 20
2.10 Carbohydrate Analysis ........... .......... .. .. .. ............................... ...... ..... ....... ... 21
2.11 Physical or Technical Effect .......................................... .... ........................ 22
Part 3: Intended Use and Dietary Exposure ............................. .... ... ... .... ............. ... 23
Part 4: Self-limiting Levels of Use ............ ............... ............................................. 24
Part 5: Experience Based on Common Use in Food Prior to 1958 ........................ 25
Part 6: Narrative ................................. ..... ........... .. ....... .......... ................................. 26
6.1 History of Consumption ...... ... ............................ ........... ........................ ....... 26
6.2 Regulatory Opinions .................... ........... .. ......... ..... ..................................... 26
6.2.1 Europe ....................................... .. ........................................................... 26
6.2.2 United States ................................... ............... .......... .............................. 27
6.2.3 Canada .... ...... .... ........ ........... .. .... .......... ..... .... ........................................ . 28
6.3 Safety Information ....................................................................................... 28
Bacillus sublilis Bss-19 GRAS 3

~l AIBMR Life Sciences, Inc.
6.3 .1 Toxicological Studies on B. subtilis strains ......... .. ....................... .... ..... 29

6.3 .2. Unpublished Acute Toxicity Study on B. subtilis Bss-19 ......... ........... 30
6.3.3. Human Studies .......................................... .... ... ............................ ......... 31
6.3.4 Opportunistic Infections ... ..... ... .. .. .......... .. ............. .............. .... .............. 32
6.4 Allergenicity .. ........... .. ... .... ..................................... .... ..... ..................... ........ 32
6.5 Past Sales and Reported Adverse Events .... .. .. ....... .. ............... ..................... 33
6.6 Basis for the GRAS Conclusion ................................................................... 33
6.6.1 Data and Information that Establish Safety .. ... .... ........ ....... .................. . 33
6.6.2 Data and Information that are Co1Toborative of Safety .................. ....... 34
6.6.3 General Recognition ............. ................ ............. .......................... .. ........ 35
6.6.4 Data and Infonnation that are Inconsistent with the GRAS Conclusion35
6.6.5 Information that is Exempt from Disclosure under FOIA ...... ............... 35
Part 7: Supporting Data and Information ... ... .......... .. .. ........... ... ............................. 36
7 .1 Data and Infonnation that are not Generally Available ............................... 36
7 .2 References that are Generally Available ................... .... ....... .. ....... .............. 36

Figures and Tables
Table 1. Genomic Average Nucleotide Identity Comparison of Bss-19 to Other Relevant

Strains ....... .............. ........... ..... .......... ............... ...... ... .... ....... .... ....... ........ ...... ... 1O
Figure 1. Manufacturing Flowchart ........... ...... .................................................. .............. 14
Table 2. Bss-19 Specifications ..................................... .......................... ....... .......... ....... 14
Table 3. Bss-19 Batch Analyses ................................. ............. .... ........ ............. .............. 15
Table 4. Bss-19 Phenotypic Resistance to Antibiotics Results ........ .... .......................... 17
Table 5. Acid Tolerance and Bile Tolerance of Bss-19 in its Vegetative State and Spore
Form ................................................... ................. .... .... ........ ... ......................... 19
Table 6. Sugar Fermentation Capacity (API 50 CH) of Bss-19 .. ..................... ............ ... 21
Table 7. FDA GRAS Notifications that Include B. subtilis Strains .. ... .. ........................... 27
Bacillus subtifis Bss- 19 GRAS 5

~ AIBMR Life Sciences, Inc.
Part 1 : Signed Statements and Certification

1.1 Submission of GRAS Notice
Danisco USA, Inc. (the notifier), an affiliate of DuPont Nutrition & Biosciences
(hereafter referred to as DuPont) is submitting a new GRAS notice in accordance
with 21 CFR Part 170, Subpa1i E, regarding the conclusion that Bacillus subtilis
Bss-19 is Generally Recognized as Safe (GRAS) for its intended use, consistent
with section 20l(s) of the Federal Food, Drug and Cosmetic Act.
1.2 Name and Address of the Notifier and Agent of the Notifier
Notifier
Elizabeth McCartney
Regulatory Affairs Specialist
Danisco USA, Inc.
DuPont Health & Biosciences
3329 Agriculture Drive
Madison, Wisconsin 53716
Tel: (608) 216-6307; Fax: (608) 395-2630
elizabeth.mccartney@dupont.com
Agent of the Notifier

Jessica Gruber, ND
Scientific and Regulatory Consultant
1425 Broadway, Suite 458
Seattle, WA 98122
Tel: (253) 286-2888
jessica@aibmr.com
1.3 Name of the Substance

The name of the substance is Bacillus subtilis Bss-19.
1.4 Intended Conditions of Use

B. subtilis Bss-19 (hereafter referred to as Bss-19) is intended to be used as an
ingredient added to foods where standards of identity do not preclude such use. It is
not intended to be added to infant formula, or any products that would require
Bacillus subtilis Bss-19 GRAS 6

additional regulatory review by USDA. The intended addition level to foods is up

°
to 1 x 10 1 CFU per serving.
1.5 Statutory Basis for GRAS Conclusion

The conclusion of GRAS status of Bss-19 for its intended conditions of use, stated
in Part 1.4 of this notice, has been made based on scientific procedures.
1.6 Not Subject to Premarket approval

We have concluded that Bss-19 is GRAS for its intended conditions of use, stated
in Part 1.4 of this notice, and, therefore, such use of Bss-19 is not subject to the
premarket approval requirements of the Federal Food, Drug, and Cosmetic Act.
1.7 Data and Information Availability Statement

The data and information that serve as the basis for this GRAS conclusion will be
available for review and copying during customary business hours at the office of
Elizabeth McCartney (DuPont Health & Biosciences, 3329 Ag1iculture Drive
Madison, Wisconsin 53716), or will be sent to FDA upon request.
1.8 Exemption from Disclosure under the Freedom of

Information Act
None of the data and information in Parts 2 through 7 of this GRAS notice are
considered exempt from disclosure under the Freedom of Information Act (FOIA)
as trade secret or commercial or financial information that is privileged or
confidential.
BacWus subtilis Bss-19 GRAS 7

./I~ AIBMR Life Sciences, Inc.
1.9 Certification of Completion
We hereby certify that, to the best of our knowledge, this GRAS notice is a
complete, representative, and balanced submission that includes unfavorable
information, as well as favorable information, known to us and pertinent to the
evaluation of the safety and GRAS status of the use of Bss-19.
(b) (4)
21 July 2020
Elizabeth McCartney Date

Regulatory Affairs Specialist
Notifier

ffl AIBMR Life Sciences, Inc.
Part 2: Identity, Method of Manufacture, Specifications,

and Physical or Technical Effect
2.1 Identification
B. subtilis is a gram-positive, rod-shaped, endospore-forming bacterium found in
the soil, on plants, in water sources, and in the gastrointestinal tract of humans. 1• 2 It
has several flagella and is highly motile. 1 While there are members of the Bacillus
genus that are known to have toxic effects in humans and animals via production of
toxins (e.g. B. anthracis, B. cereus), B. subtilis has a long history of safe use for
human consumption as will be detailed in Part 6. 3
Dupont ' s Bss-19 is also known as B. subtilis BS771 l. Additionally, DGCC12972
is used as the internal identification within the DuPont Global Culture Collection.
2.1.1 Taxonomy of Bss-19

Dupont's B. subtilis inaquosorum strain Bss-19 has been identified according to
standard taxonomic guidelines. The taxonomic lineage of the strain is:
Kingdom: Bacteria
Phylum: Firmicutes
Class: Bacilli
Family: Bacillales
Genus: Bacillus
Species: Bacillus subtilis
Strain: inaquosorum strain Bss-19
2.1.2 Genetic Identification of Bss-19

The whole genome ofBss-19 was sequenced, assembled, and included a single 16S
rRNA gene copy. When compared to publicly available sequences, the 16S rRNA
copy of Bss-19 was found to be most closely related to the type strain B. subtilis
subspecies inaquosorum KCTC 13429 (99.93% identical).
Whole genome sequencing and assembly ofBss-19 revealed seven single nucleotide
variations between the assemblies when compared to publicly available B. subtilis
inaquosorum strain DEl 11 sequence. Four of these changes were confirmed by
PCR. Two of the single nucleotide variations resulted in amino acid changes
indicating that these strains do not have identical genomes.
Genomic average nucleotide identity (ANI) comparison of the whole draft genome
sequence of strain Bss-19 to relevant, closely related strains: DE 111, KCTC 13429
(the type strain for the subspecies inaquosorum), and strain 168 (the type strain for

. . AIBMR Life Sciences, Inc.
the closely related subspecies B. subtilis subsp. subtilis) was performed. ANI values
above 95% are considered representatives of members of the same species. ANI
calculations, shown in Table 1, indicate Bss-19 is of the same species as DE 111 and
KCTC 13429; but that inaquosorum may be considered a separate species from the
subtilis subspecies. DuPont states that ANI is calculated using two factors to
produce a third. Overall percent identity of the portions of the sequence that align
(ANI) and fraction of the two genomes which do align (coverage) are multiplied to
produce the Hadamard product. The Hadamard product is usually a smaller percent
identity than the initially reported percent identity unless coverage is complete for
both genomes.
Table 1. Genomic Average Nucleotide Identity Comparison of Bss-19 to Other

Relevant Strains
B. subtilis inaquosorum B. subtilis subtilis
strain strain strain
DElll KCTC 13429 168
Coverage 99.84% 93.86% 84.52%
Percent identity (ANI) 99.98% 98 .83% 93 .00%
Hadamard product 99.83% 92.77% 78.61%
DuPont states that whole genome sequences of Bss-19, DElll, and KCTC 13429
aligned using the Mauve Progressive Alignment4 tool indicate strong conservation
between Bss-19 and DEl 11, but KCTC 13429 appears to contain unique regions
ranging in size from 2 to~40 kb .
2.2 Manufacturing
2.2.1 Good Manufacturing Practice
All production steps of Bss-19 are consistent with current Good Manufacturing
Practice (cGMP) guidelines in an FDA regulated and inspected facility.
2.2.2 Raw Materials

Raw materials used in the production of DuPont's Bss-19 are of appropriate food
grade and are suitable to the application to produce the final food grade product.
2.2.3 Manufacturing Narrative and Flowchart

Master Seed
The source organism used is Bacillus subtilis Bss-19.
Bacillus subtilis Bss-1 9 GRAS JO

. . . . AlBMR Life Sciences, Inc.
DuPont takes great care to ensure the quality of bacteria fermentation products.
These quality control processes begin with the identification, storage, and handling
of the bacteria seed stocks.
A Master Seed repository is maintained for each of the bacterial strains at the
DuPont Global Culture Collection (DGCC) in Niebiill, Germany. The repository is
a collection of purified, tested, and qualified Master Seed stocks derived from single
strain isolates stored at -180°C in liquid nih·ogen to maintain long term cell viability.
Each seed lot in the culture bank is fully characterized to ensure the identity of the
seed strains.
Whole genome sequencing is conducted to establish the identity of each bacteria to
the genus, species, and strain level prior to preservation. The microbiological quality
of the Master Seeds is determined by testing for microbiological contamination at
the DGCC. These identity and purity specifications are absolute acceptance criteria
for the Master Seeds. If a Master Seed vial lot fails any of the required tests, the lot
is placed on Quality Control (QC) hold to prohibit use and the lot is subsequently
destroyed.
Working seed
Working seeds are prepared under controlled conditions from master seed stock
maintaining effective acceptance criteria at DGCC. All Working Seeds are prepared
under controlled conditions from Master Seed stock meeting established acceptance
criteria and each new lot of Working Seeds is held in quarantine pending QC testing
(strain identity and purity as described for the Master Seeds) and release. If the
Working Seed vial lot fails any of the required tests, the lot is placed on QC hold
and destroyed. Qualified, tested Working Seed stocks are stored at -80°C until used
in production fermentation.
The use of tandem Master and Working seed inventories reduces the risk of genetic
drift over time due to excessive sub-culturing of strains and ensures the integrity of
the strain collection. All steps in the preparation of Master and Working seed are
documented in a specified database, allowing h·aceability of every seed preparation
down to each single batch of raw material used.
Fermentation process
The fermentation begins by withdrawing one of the working seed vials and scaling
up via a series of fermentations until a commercial size batch is complete.
The fermentation media contains a buffer system to minimize pH shifts. The pH
profile for each batch is monitored against a standard to ensure repeatability. The
pH in the fermenter is monitored on digital display and on recording cha1is. By
consulting these charts, the growth and sporulation characteristic of a given
fermentation can be determined.
Bacillus subtilis Bss- 19 GRAS 11

After fermentation is complete, an additional aeration step is performed in the

fermenter. Airflow is increased and back pressure decreased for a specified amount
of time. The fermenter is then normally cooled to stop the fermentation at a specified
time. Cooled fermentate is pumped through a continuous flow centrifuge and the
bacterial spores are concentrated. The bacterial spore concentrate is pumped to a
spray dryer where it is atomized and dried. After spray drying, the bacterial spore
concentrate is bulk packaged with lot control and traceability, then stored until QC
release against specified criteria.
The fem1entation production process is a closed system with no product exposure
from seed inoculation to cell harvest. Prior to each fermentation batch, all mixing
tanks, lines, fermenter, centrifuge, and spray dryer are cleaned via automated clean
in-place systems. Systems are then either steamed or chemically sanitized prior to
product contact.
Packaging
Bulk packaging of the product is carried out in a controlled environment within the
DuPont Rochester facility.
HEPA filter is used in the packaging room for high perfonnance as the final filter
for particulate removal when clean air is required.
Final packages are heat sealed before passing through a metal detection x-ray
system.
Quality Systems
The DuPont Rochester plant has fully implemented HACCP plans, Standard
Operating Procedures and Quality Control programs to ensure the quality of each
product. A quality control laboratory is maintained on site. Quality control
personnel are qualified by training and experience to test products and to release
product based on specifications. In addition, a third-party approved laboratory with
ISO 17025 certification performs QC testing for DuPont under contract.
The Quality Control unit utilizes an SAP computer quality control system for the
specification, quality control data entry and product release. No product can be
released for use without acceptance by the Quality Control unit, according to
specified acceptance criteria.
Each bacteria fermentation product must meet specifications and must have a
confirmation of identity through a PCR (polymerase chain reaction) test method
based on strain specific primers for release of the product. Microbiological testing
is performed by trained QC microbiologists in the Rochester plant laboratory and
an approved external laboratory using standard methods.

Cleaning and quality testing of the process rooms and equipment are under the
control of Manufacturing and Quality Assurance, following the established SOPs.
Room access is controlled by appropriate signage, and additional protective
gowning must be worn in processing rooms where product is potentially exposed.
Operator sign-off for cleaning, sanitation and testing are required on proper
documentation.
Process rooms are segregated from other manufacturing areas with appropriate
closures. Room air quality is controlled via HEPA air filtration of incoming air and
maintenance of positive pressure in the process rooms relative to adjacent
processing areas. HEPA filtration operation is monitored for performance; air
quality is monitored monthly by Quality Assurance.
Rooms and equipment used in manufacturing are approved for production only after
cleaning, sanitization, and inspection. Prior to qualification of the process room for
production, as specified in the appropriate SOP, the Spray Dried room is sprayed
from ceiling to floor with 145-l 60°F water. All clean, out of place equipment
having any product contact surfaces is thoroughly scrubbed / foamed with a neutral
detergent cleaner, rinsed with cold water, and sanitized with an acid-based sanitizer.
The floor is sanitized with acid-based sanitizer.
Batch records are maintained as per Standard Operating Procedures and are
provided to Quality Assurance for each lot produced. Quality Assurance is
responsible for batch ticket review. The flowchart of the manufacturing process is
shown in Figure 1.

. . . . A1BMR Life Sciences, Inc.
Manufacturing Process Step

Process Controls Confirmation
GLPs Approved Seed Culture QC Testing
HACCP,GMPs Seed Scale-up
HACCP.GMPs Commerical Fermentation
HACCP,GMPs Culture Concentration QC Testing
HACCP.GMPs Culture Spray Dried QC Testing
H ACCP,GMPs Metal Detection Standards Testing
HACCP.GMPs Culture Packaging
QC Release and Storage QC Testing
Figure 1. Manufactu ring Flowchart
2.3 Specifications
The specifications for the food-grade product Bss-19, along with the specification
methods, which have been validated for their stated purpose, are listed in Table 2
below.
Ta bl e2. Bss-19 s,pecifications

Te_sted Parameters Spe~ification Method
Viable cell count (CFU/g) > 2.25 X 10 11 In-house method
Color 1 White to cream Visual Inspection
Form 1 Freeze-dried powder Visual Inspection

Heavv Metafs2
Arsenic (oom) < 1.0 ppm AOAC 984.27
Cadmium (ppm) < 0.2 Porn AOAC 984.27
Mercury (ppm) < 0.05 ppm EPA 7471
Lead (ppm) < 0.5 ppm AOAC 984.27
Mi~robioloc:1ical Tests
Coliform (CFU/g) < 10 AOAC 991.14
Escherichia coli (CFU/g) < 10 AOAC 991.14
Salmonella Negative in 50 g AOAC Rl-121501
Listeria Negative in 25 g FDA BAM Chaoter 10
Staph ylococcus coag+ (CFU/g) < 10 FDA BAM Chapter 12
B. cereus (CFU/g) < 10 AOAC 980.31
Molds and Y east3 < 100 CFU/g USP 41-NF 36
..
Abbreviations: CFU, colony forming unit; AOAC, Assoc1atton of Official Analytical Chemists; EPA, Environmental
Protection Agency; FDA BAM, Food and Drug Adm inistration's Bacteriological Analytical Manual; ppm, parts per
million
1 Internal Specification, not reported on CoA
2 Based on a1mual surveillance testing, not generally reported on CoA
3 Tested on bulk intermediate powder, not genera lly reported on CoA
2.3.1 Batch Analysis

Production conformity and consistency of DuPont' s Bss-19 are tested in production
lots. Batch analyses of three non-consecutive lots are shown below and are
reasonably consistent and met the product specifications for marker compounds,
microbial analyses, and heavy metals (see Table 3).
Ta bl e 3 Bss- 19 Bate h Ana1yses

Lot NoJDate of Manu facture
Tested Parameters Sp_ecificatian 1493413902 1493441559 1493438028
03/29/2019 04/11/2019 04/19/2019
Viable cell count (CFU/g) ::'. 2.25 X 10 11 4.03 X 10 1I 3.63 X 10 11 3.73 X 10 11
Microbiological Tests
Coliforms (CFU/g) < 10 < 10 < 10 < 10
Escherichia coli (CFU/g) < 10 < 10 < 10 < 10
Salmonella spp. ND in 50 g Negative/SO g Negative/SO g Negative/50g
Listeria spp. ND in 25 g Negative/25 g Negative/25g Negative/25g
Staphylococcus aureus < 10 < 10 < 10 < 10
(CFU/g)
B. cereus (CFU/g) < 10 < 10 < 10 < 10
Heavy Metals
Arsenic (ppm) < 1.0 0.05 0.05 0.06
Cadmium (ppm) < 0.2 0.03 0.02 0.02
Mercury (ppm) < 0.05 < 0.02 < 0.02 < 0.02
Lead (ppm) < 0.5 < 0.010 < 0.010 < 0.010
. .
Abbrevwtwns: CFU, colony forming umts; ND, not detected; ppm, parts per m1lhon
..

2.3.2 Residual Pesticide Analysis

In accordance with standard operating procedures, DuPont is committed to annual
surveillance testing of Bss-19 for pesticide residues. All lots in Table 3 of Bss-19
were analyzed using AOAC 2007.01 for the presence of hundreds of residual
pesticides by an independent laboratory. All lots were free of all pesticides tested
and complied with the product specifications.
2.3.3 Shelf-Life Stability

A long-term stability study for Bss-19 is cun-ently underway.
2.4 Antibiotic Resistance

Resistance to therapeutic antibiotics in microbial pathogens is currently considered
one of the greatest challenges in medicine and public health, as some infectious
diseases may become virtually unh·eatable if they become non-respondent to their
current therapies. Antibiotic resistance may be classified into two types;
• intrinsic/natural (when resistance is inherent to a bacterial species, and is a
trait generally shared by all members of that species), and
• extrinsic/acquired (when a strain of a typically susceptible species is resistant
to a given antimicrobial drug).
Extrinsic/acquired resistance can occur either from the gain of exogenous DNA or
mutation of indigenous genes. 5, 6 The gain of exogenous DNA occurs through
horizontal gene transfer (HGT) via transformation, transduction or conjugation and
many of the antibiotic resistance genes are carried on mobility elements such as
plasmids, transposons, or phages.7•8 While intrinsic resistance likely presents a very
low risk of dissemination, exh·insic/acquired resistance, especially when the
relevant genes are associated with mobile genetic elements, can be transferred to
pathogens or other commensal bacteria. 7• 9
It is generally recommended that resistance to antibiotics be assessed in all probiotic
strains prior to marketing. 5• 10- 13 Antibiotic resistance is a complex phenomenon, in
which microbial genetics and environmental stimuli both play an imp01iant role.
Assessing resistance both phenotypically and genotypically is generally
recommended. As detailed below, antibiotic susceptibility ofBss- 19 was evaluated
using both approaches.
Phenotypic evaluation of antibiotic resistance involves testing the capacity of a
microorganism to survive in a medium containing different concentrations of
antibiotics. Whereas most microorganisms can survive at low concentrations of
many antibiotics, resistance is defined as the capacity to grow at antibiotic
concentrations similar to those reached in the human body during therapeutic
intervention.

,l AIBMR Life Sciences, Inc.
Fallowing EFSA recommendations and guidelines, DuPont assessed the phenotypic

susceptibility of Bss-19 to the antibiotics detailed in the guidelines for Bacillus
species, namely gentamycin, kanamycin, erythromycin, clindamycin, tetracycline,
streptomycin, vancomycin, and chloramphenicol. 14 One additional antibiotic,
Ampicillin, was also tested although it is not a requirement per the EFSA guidelines
for Bacillus species. The EFSA guidelines define a bacterial strain as sensitive or
susceptible to an antibiotic when it is inhibited at a concentration of a specific
antimicrobial equal or lower than the established cut-off value for that particular
compound.
The assays on Bss-19 were performed using the ISO l 0932 IDF223 method and
VetMIC Lact-1 and 2 micro-dilution plates that include all antibiotics recommended
by EFSA. All MIC values were below or equal to the microbial break points defined
for Bacillus species except for chloramphenicol. Results are shown in Table 4 and
indicate that Bss-19 is phenotypically sensitive to all antibiotics included in EFSA 's
guidelines for the B. subtilis species except for chloramphenicol.
Table 4. Bss-19 Phenotypic Resistance to Antibiotics Results

MIC (mg/t.}
Antibiotic;: Bss-19 B. subtilis Assessment
(a6served) Breakpoinfss
Ampicillin 0.06 n.r. Sensitive
Clindamycin 2 4 Sensitive
Chloramphenicol 16 8 Resistant
Erythromycin 0.5 4 Sensitive
Streptomycin 4 8 Sensitive
Gentamycin 1 4 Sensitive
Kanamycin 4 8 Sensitive
Tetracycline 0.25 8 Sensitive
Vancomycin 1 4 Sensitive
.. . .
MlC = Mm1murn Inh1b1 tory Concentration; n.r. = not reqmred (accordmg to EFSA gu1delmes for B. subtilis) 14
Genotypic evaluation of antibiotic resistance is a procedure in which the whole

bacterial genome (chromosome and peripheral genetic elements, if any) is screened
for putative genes of antibiotic resistance, as described in genetic databases. It is
therefore a complementary procedure to the phenotypic assessment, in which the
main objective is to discard the potential of transferring putative genes of antibiotic
resistance to other microbes.
Comparison of all annotated Bss-19 protein sequences to the Comprehensive
Antibiotic Resistance Database (CARD) 15 revealed a putative homolog to a
chloramphenicol acetyltransferase resistance protein (53% identity over 100% of
query length). This gene was also found in both the B. subtilis inaquosorum DEl 11
and B. subtilis inaquosorum KCTC13429 genomes. Three types of HGT were

A AiBMR Life Sciences, Inc.
evaluated by DuPont and included: conjugative plasmids, transposases, and

prophage/bacteriophage elements. A search of the Bss-19 draft genome sequence
versus the ISFINDER database 16 of known mobility element sequences revealed no
mobility loci within 15 kb of the putative chloramphenicol resistance gene.
Additionally, no intact or potentially active phage or prophage genomes were
identified in the assembled Bss-19 genome sequence.
The lack of the presence of known mobility elements near the chloramphenicol
resistance gene combined with the absence of active phage or prophage genomes
suggests that acquired antibiotic resistance for the strain is not a concern.
Additionally, while the observed MIC for Bss-19 was just above the cut-off value
established by EFSA, one two-fold dilution above the EFSA cut-off value is still
generally considered acceptable. This is because of the technical variation of the
phenotypic method applied to determine antibiotic susceptibility. There is a certain
amount of technical variability in all phenotypic antibiotic-resistance testing.
Guidelines for this testing allow for this normal (technical) variation around the
mean. The phenotypic test perfo1med is generally based on two-fold broth dilutions.
There is precedent for accepting levels that exceed the MIC cut-off by a single two
fold dilution due to normal variation around the mean; for example EFSA's
"Scientific Opinion on the safety and efficacy of Oralin®(Enterococcus faecium) as
a feed additive for calves for rearing, piglets, chickens for fattening, turkeys for
fattening and dogs" , 17 in which the Oralin®'s MIC value exceeded the MIC cut-off
for kanamycin by a single two-fold dilution, was considered to be within normal
variation and did not raise concerns for safety by EFSA.
2.5 Genomic Analysis for Virulence and Pathogenicity

The genome ofBss-19 was screened using the Database of Bacterial ExoToxins for
Human Health (DBETH) 18 which revealed a single hit (70% identity over 90% of
query length) to a hemolysin protein (HL Y3 _ BACCE). However, the Bss-19 gene
encoding this protein is highly conserved across the genus including many strains
known to not be pathogens. Additionally, as discussed in Part 2.7, Bss-19 showed
no hemolysis when analyzed in vitro.
A comparison of all Bss-19 protein sequences to the virulence factor database
(VFDB) 19 revealed no significant matches, indicating that Bss-19 does not contain
any detected virulence factors.
2.6 Resistance to Gastric Acidity and Bile Salts

In many cases, probiotic microorganisms should be viable upon reaching the
gastrointestinal tract. Some important traits that are believed to be relevant for
surviving the passage through the gastrointestinal tract are tolerance to the acidic
environment of the stomach and tolerance to the concentration of bile salts found in
the small intestine. The extremely acidic environment of the stomach (pH of
Bacilfus subti/is Bss-19 GRAS 18

~ ..LBMR Life Sciences, Inc.
approximately 2.0- 3.0) kills the majority of potentially pathogenic bacteria,

preventing infection.
For assessment of Bss-19, a modified gastric juice was utilized to simulate contact
with a moderately acidic stomach fluid environment. Bile tolerance was estimated
by determining the % recove1y on bile containing agar medium compared to a non
bile containing control medium.
Culture was obtained from seed vials, inoculated into strain specific medium, and
grown overnight. An aliquot of the overnight broth culture was pelletized, washed,
and resuspended. An aliquot of the resuspended pellet was mixed with tempered
gastric juice (hydrochloric acid and pepsin (0.32%) and pH 3.5). An aliquot was
immediately diluted and plated in TSA agar with and without 0.3% ox-gall bile salt
for a TO control and a bile test result. The balance of sample in gastric juice was
incubated for one hour at which time the final aliquot was taken for Tl plating using
the same TSA media. Plates were allowed to solidify and incubated at 32°C under
aerobic conditions for 48 hours . The results are shown in Table 5.
Table 5. Acid Tolerance and Bile Tolerance of Bss-19 in its Vegetative State and
S.pore Farm
lntemal Acid tolerance Bile tolerance Date Tested
Identification
Vegetative state DGCC12972 * <* I 0% <* I 0% 5/13/2019
Spore form DGCC12972 * **** I 100% *** I 88.4% 5/30/2019
*DGCC!2972 (DuPont Global Culture Collection) 1s used as the Internal Ident1ficat1on for Bss-19
Acid Tolerance rating

**** Excellent (>90% survival in hydrochloric acid and pepsin, 0.32% (wt/v) at pH 3.5)
*** Very Good (80- 90% suivival)
** Good (70-79% survival)
* Fair (<69% survival)
Bi le Tolerance Rating
*·* ** Excellent (>90% survival in 0.3% bile salt containing medium)
** * Very Good (80-89% suivival)
** Good (70-79% survival)
* Fair (<69% survival)
Bss-19 in the vegetative state exhibited 0% survival following exposure to a low pH

solution and 0% survival in bile salt solutions. Bss- 19 in its spore form exhibits
>90% survival in a low pH solution and >80% survival in a bile salt solution. These
results suggest the spore form is necessary for survival through the gastrointestinal
tract.
2.7 Hemolysis
Cultures of the strain were grown overnight in strain specific medium and
temperature. An aliquot of the overnight culture was streaked onto prepared blood

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agar plates and incubated at a strain specific temperature for 18 and 24 hours.
Results showed that Bss-19 did not promote hemolysis when cultured on Sheep's
blood agar plates.
As discussed in Part 2.2, genomic analysis of Bss-19 revealed a single hit to a
hemolysin protein (HL Y3 _ BACCE). However, the Bss-19 gene encoding this
protein is highly conserved across the genus including many strains that do not show
pathogenic activity. This is supported by the absence of hemolysis observed when
the strain is grown on blood agar.
2.8 Biogenic Amine Formation

Some species and/or strains of lactic acid bacteria are able to produce biogenic
amines (organic, basic, nitrogenous compounds formed mainly by the
decarboxylation of amino acids), likely for use as metabolic energy and/or to
increase acid resistance. 20 These amines are present in a wide range of foods (e.g.,
fermented food products), and although they are involved in many natural
physiological processes, consuming large quantities of these amines can have
undesirable consequences in some individuals. For example, if they are not properly
biotransformed in the body, they can cause release of adrenaline/noradrenaline,
cause gastric acid secretion, increased cardiac output, heart rate, and blood pressure,
migraines, and increased blood sugar. 20 Biogenic amine formation in fermented
foods has been reviewed by EFSA (20 11) 21 and Spano (2010). 20 Histamine and
tyramine are considered the most concerning with regard to food safety. 21
Generally, detection of strains possessing amino acid decarboxylase deaminase
activity is helpful to aid in mitigating the accumulation of these amines in food
products. Per assessment, Bss-19 did not contain histidine decarboxylase-encoding
genes or tyrosine decarboxylase-encoding genes in its genome. Hence, it is unlikely
that this strain can produce histamine or tyramine.
2.9 D(-)/L(+)-Lactic Acid Production

Bss-19 produces lactic acid (lactate) from the fermentation of carbohydrates.
Lactate exists in two forms, a dextrorotary enantiomer (D-lactate) and a levorotary
enantiomer (L-lactate). In humans, over 99% of lactate found in the blood is L
lactate. Testing D-lactate production by food microorganisms has been historically
recommended likely because until relatively recently, it was believed that humans
had a poor capacity for metabolizing D-lactate. 11 Some lactic acid bacteria as well
as several other members of the intestinal microflora produce a mixture of L- and
D-lactate. 22 More recent studies have shown that much of the human gut microbiota
produces D-lactate with no evidence of D-lactic acidosis, and in fact, humans are
able to metabolize this isoform.23 -29 D-lactate accumulation may only occur in cases

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of impaired D-lactate metabolism and/or in subjects with a disturbed gastrointestinal

function following bowel resection or Short Bowel Syndrome (SBS). 25 • 29 · 32
Cultures of the strain were grown overnight in strain specific medium and
temperature. An aliquot of overnight broth culture was pelletized, and the
supernatant was retained. The supernatant was inactivated at 80°C for 15 minutes
and diluted to achieve the desired total lactic acid concentration range. D(-)/L(+)
lactic acid detection was pe1formed using a calorimetiic measurement of lactate
dehydrogenase activity for the respective isomers. Total lactic acid and isomer
specific measurements were determined relative to control samples. Bss-19
produced an average of 100% of the L(+)-lactic acid isomer and 0% of the D(-)
lactic acid isomer.
2.1 O Carbohydrate Analysis

Bss-19 capability to use and ferment different sugars as carbon sources was assessed
following the API 50 CH system (Biomereux, France). Results are shown in Table
6.
Table 6. Sugar Fermentation Capacity (API 50 CH) of Bss-19

Substrate Growth Substrate Growth Substrate Growth
Control - Inositol + Melezitose -
Glvcerol + Mannitol + Raffinose +
Erythritol - Sorbitol + Starch +
a-methyl-D-
D-Arabinose - Mannoside
- Glycogen +
a-methyl-D-
L-Arabinose + Glucoside
- Xylitol -
N-
D-Ribose + Acetylglucosamine
- Gentiobiose -
D-Xvlose - Amvgdaline + D-Turanose +
L-Xvlose - Arbutine + D-Lvxose -
Adonidol - Esculin + D-Tagatose -
Beta Methyl-D-
- Alicin + D-Fucose -
Xyloside
Galactose - Celiobiose + L-Fucose -
Glucose + Maltose + D-Arabitol -
Fructose + Lactose - L-Arabitol -
Mannose + Melibiose + Gluconate -
2-Keto-
Sorbose - Sucrose + gluconate
-
5-Keto-
Rhamnose + Trehalose + gluconate
-
Dulcitol - lnulin +

2.11 Physical or Technical Effect

Bss-19 is not intended to produce any physical or other technical effects that are
relevant to the safety of the ingredient.

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Part 3: Intended Use and Dietary Exposure

For the purpose of this GRAS notice, Dupont's Bss-19 manufactured in accordance
with current GMP, is intended to be used as an ingredient added to foods where
standards of identity do not preclude such use. For example, it maybe be used in
yogurt and other dairy products, soy products, beverages, chewing gum,
confectionary, snacks, and other foods. It is not intended to be added to infant
formula, or any products that would require additional regulatory review by USDA.
°
The intended addition level to foods is up to 1 x 10 1 CFU per serving (which is
similar to levels of lactic acid bacteria found in traditionally fermented food
products). 33
The addition of Bss-19 to some foods may be substitutive with regard to other
GRAS B. subtilis strains' intended uses (e.g. GRN 831 and 905), or with regard to
traditional uses of B. subtilis. However, uses in other foods may be considered more
novel and additive with regard to exposure. Several older publications were located
that looked at dietary patterns of Americans by analyzing the number of servings of
foods consumed in a day. A publication from the USDA's Center for Nutrition
Policy and Promotion (October 2000) states that men aged 51 and older consume
the largest number of servings of food per day, at 18.2 servings/day. 34
Comparatively, women aged 19-24 consumed the least, at 12.5 servings/day. This
data came from detailed 14-day food diaries from 5,752 adults in the 1992-1994
time period. Millen et al. (2005) used 24-hour dietary recall and diet history
questionnaire data from the Eating at America's Table study ( 1997-1998) to analyze
the mean number of servings per day consumed of food guide pyramid food groups
by adults. 35 There were 497 women and 436 men that completed the study. The
results (from the study's Table 1) suggest that the mean intake for men was
approximately 27.8 servings per day and for women was 19.5 servings per day.
Using a most conservative estimation of consumption, if 100% of food servings
°
contained Bss-19 at the maximum concentration of 1 x 10 1 CFU per serving,
highest consumers (men) would be exposed to approximately 1.82-2.78 x 10 11
CFU/day. Using 70 kg as a standard body weight, this is equivalent to 2.6-4.0 x 109
CFU/kg bw/day). This estimation is considered extremely conservative, as
realistically, most foods will not contain Bss-19 due to the standards of identity of
many foods, the fact that it will not be added to foods requiring additional USDA
regulatory review, market share limitations, limited food matrix viability, and the
fact that the ingredient will likely be "invisible" to many consumers, who may
realize they are consuming a fermented food but likely will not be aware of the
specific strain that they are consuming, reducing the likelihood that only food
products containing this strain will be chosen and consumed. If a more realistic (but
still highly conservative) estimate is used that 50% of food servings will contain the
maximum intended use level ofBss-19, highest consumers (men) would be exposed
to approximately 9.1 x 10 10 to 1.4 x 10 11 CFU/day (using 70 kg as a standard body
weight, this is equivalent to 1.3-2.0 x 109 CFU/kg bw/day).

Part 4: Self-limiting Levels of Use

There are no known inherent self-limiting levels of use.

Part 5: Experience Based on Common Use in Food Prior

to 1958
The GRAS conclusion for Bss-19 is based on scientific procedures, and thus,
experience based on common use in food prior to 1958 is not considered pivotal
information. Nevertheless, the historical use of foods fermented with B. subtilis is
extensively discussed in Section 6.
Bacillus subti/is Bss- 19 GRAS 25

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Part 6: Narrative
6.1 History of Consumption
B. subtilis has a long history of human consumption, especially in fermented foods
in Asia and Africa. Hong et al. (2005) describes at least nine probiotics on the
market containing B. subtilis that are intended for human consumption, many of
which have been on the market for decades without safety concems. 36 Typical levels
ranged from 1 x 106 to 1 x 10 9 CFU/serving. 36
B. subtilis is well known for its use in the traditional Japanese fermented soybean
food called natto, which has a bacterial concentration reported as approximately 1
x 108 CFU/g. 1, 36 Consumption of a 100g serving of natto containing this
concentration of bacteria is equivalent to consumption of approximately 1 x 10 10
CFU/serving. B. subtilis natto is recognized as FOSHU (Food For Specified Health
Use) by the Japanese Ministry of Health, Labour, and Welfare.37
B. subtilis is also listed in the inventory published by the International Dairy
Federation (originally a collaboration with the European Food and Feed Culture's
Association) documenting microbial species with technological beneficial roles in
fermented food products, specifically as relates to use in soy (natto), emphasizing
the species' long history of use. 38 , 39
6.2 Regulatory Opinions
6.2.1 Europe
EFSA has developed an approach to safety assessments of microorganisms called
Qualified Presumption of Safety (QPS). QPS generically assesses the safety of
taxonomic groups or units (e.g. a bacterial species) independent of any particular
pre-market authorization process. Any strain of microorganism, the identity of
which can be unambiguously established and assigned to a QPS group, does not
need to undergo further safety assessment by EFSA other than satisfying any
qualifications specified in the QPS assessment. QPS is generally not based on a
particular intended use unless stated in a specific qualification. Microorganisms not
considered suitable for QPS remain subject to full safety assessments. The first QPS
list was established in 2007. 3 A full evaluation of the QPS list is undertaken every
3 years and results are published as Scientific Opinions (the next mandated Opinion
was published in December 2019), while the list of microorganisms is maintained
and re-evaluated approximately every 6 months to include new notifications to
EFSA, and published as Panel Statements (the most recent Panel Statement was
published in June of 2020 and includes research published through December
2019. 40 B. subtilis was granted QPS status in the first EFSA QPS publication in
2007, based on the substantial body of knowledge available on the species. However
because some species within the Bacillus genus possess toxigenic traits, a QPS

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qualification for this species is the absence of toxigenic activity. 3 The other
qualification is that the individual strains should not harbor any acquired
antimicrobial resistance genes to clinically relevant antimicrobials. B. subtilis
remains on the most recent EFSA QPS lists.40 , 4 1
6.2.2 United States
6.2.2.1 FDA GRAS

In the US, companies can notify FDA of their conclusion of GRAS status for a
particular bacterial species/strain or ingredient on an individual basis, and for
specific intended uses. It was estimated in 2009 that approximately 40% of food
enzymes marketed in Europe were produced by bacterial/fungal recombinant
strains, and vitamins, amino acids, and polysaccharides are also obtained from
recombinant strains. 42 Fifteen GRAS notices related to B. subtilis strains (mainly as
recombinant strains utilized to isolate enzymes) are listed in FDA's GRN inventory.
Of these, 14 have received the no questions letter from FDA, one was ceased to be
evaluated at the notifier' s request (this was actually a notice for B. subtilis itself, and
the reason for requesting that FDA cease to evaluate is unknown). A brief summary
of these notifications is shown in Table 7.
Table 7. FDA GRAS Notifications that Include B. subtilis Strai ns

FDAGRN Date of
Strain Description Comments
Numl;>er Closure
Pullulanase derived from B.
September
20 subtilis carrying a gene encoding FDA had no questions.
1999
pullulanase from B. naf(anoensis
Pectate lyase enzyme preparation from B. January
114 FDA had no questions.
subtilis 2003
Pullulanase enzyme preparation from B.
December
205 subtilis expressing the pullulanase gene FDA had no questions.
2006
from B. acidopu/lulyticus
Branching glycosyltransferase enzyme
preparation from B. subtilis expressing a June
branching glycosyltransferase gene 2009
from Rhodothermus obamensis
1,4-a-glucan branching enzyme
preparation from B. subtilis strain 168 September
expressing the glucan branching enzyme 2012
gene from Aquifex aeolicus strain VF
Asparaginase enzyme preparation
February
476 prnduced by genetically modified B. FDA had no questions.
2014
subtilis
April At the notifier's request, FDA
562 B. subtilis
2015 ceased to evaluate the notice.

Lactase from Bifidobacterium November

bifidum produced in B. subtilis 2015
October
592 P-glucanase from B. subtilis FDA had no questions.
2015
p-galactosidase enzyme preparation November
from B . circulans produced in B. subtilis 2016
Subtilisin from B. February
amyloliquefaciens produced in B. subtilis 2018
Maltogenic amylase from Geobacillus June
stearothermovhilus produced in B. subtilis 2018
Maltogenic alpha-amylase from B. July
751 FDA had no question
stearothermophilus produced in B. subtilis 2018
October
831 B. subtilis DEl 11 FDA had no questions.
2019
June
905 B. subtilis DSM 32444 FDA had no questions.
2020
6.2.2.2 Code of Federal Regulations

There are three regulations in the 21 CFR for enzyme preparations allowed in foods,
derived from nonpathogenic/nontoxigenic B. subtilis strains, as follows:
• 21 CFR 173.115 Alpha-acetolactate decarboxylase enzyme from
recombinant B. subtilis;
• 21 CFR 184.1148 Carbohydrase enzyme from B. subtilis;
• 21 CFR 184.1150 Protease enzyme from B. subtilis.
Of note, a number of B. subtilis strains are registered with the Environmental
Protection Agency (EPA) as microbial pesticides for various uses. 43 EPA describes
B. subtilis as "a ubiquitous bacteria commonly found in various ecological niches
including soil, water, and air which does not have a history of pathogenicity from
contact in the environment".43
6.2.3 Canada
A number of products containing B. subtilis are approved to be marketed under the
Natural Health Products Regulations of Health Canada, including products
containing B. subtilis strain DEl 11 (NPNs 80077102 & 80080178) and B. subtilis
strain RO 179 (NPN s 80045131, 80051112, 80054028, etc.).
6.3 Safety Information

Toxicological studies have been published on various strains of B. subtilis and are
summarized in subpart 6.3.1. Additionally, human studies on the strain of closest
similarity, B. subtilis DEl 11 and other B. subtilis strains are discussed in subpart
6.3.3. While no published human or toxicological studies were located for B. subtilis
Bacillus subtilis Bss-1 9 GRAS 28

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Bss-19 specifically, an unpublished acute oral toxicity study was performed on the
strain and is summarized in subpart 6.3.2. The studies reviewed do not suggest any
concerns related to the safety of the strain.
6.3.1 Toxicological Studies on 8. subtilis strains

Zhang et al. (2013) studied B. subtilis strain Tpb55 in an acute gavage toxicity study
and a maximum tolerable dose study in mice and rats, respectively, in which animals
were observed for 14 days after treatment. 44 The LDso was determined to be greater
than 5000 mg/kg in both the mice and rats, as no deaths occuned and in addition no
"symptoms of poisoning", or abnormal anatomic stmctures were observed.
Similarly, no increase in the incidence of micronuclei or chromosomal aberrations
occurred in in vivo mouse assays up to the highest dose tested (2500 mg/kg bw/day
for two days or five days in a bone marrow polychromatic erythrocyte micronucleus
study and a primary spermatocyte chromosomal aberration study, respectively). The
° °
spore content was 3 x 10 1 CFU/g (7.5 x 10 1 CFU/kg bw/day) in all of the studies.
Two toxicity studies published in Korean and Chinese, respectively, were identified
and the translated abstracts are described below. Both studies were also described
in GRN 831 on B. subtilis DEll 1, which received the FDA no questions letter.
Kyoung-Hoon et al. (2015) administered a single oral dose of B. subtilis JNS to
mice at 2000 mg/kg bw followed by observation for 14 days. 45 The authors reported
that no significant change in general conditions, mortalities, body weight changes,
clinical signs, autopsy findings, or presence of gross lesions were observed.
Nakamura et al. (1999) performed a 90-day subchronic toxicity study in both sexes
of F344 rats by feeding of CRF-1 pellet diet containing 0%, 0.18%, 0.55%, 1.66%
and 5% B. subtilis gum (strain and CFUs of B. subtilis were not specified). 46 Five
groups consisted of 10 males and 10 females each whereby rats were randomly
allocated. No animals died during the administration period and there were no
differences in body weights or food intakes among groups of either sex. Kidney
weight was significantly increased in both sexes in groups given concentrations of
1.66% or more B. subtilis, but these increases were slight and serum biochemistry
and histopathology did not show any toxicological effects. The authors concluded
that these findings indicated that the treatment of B. subtilis gum in the diet for 90
days does not exert toxicity in rats even at the highest dose tested.
Sorokulova et al. (2008) described a number of studies on B. subtilis VKPM B2335
(BS3). 47 Groups of 10 BALB/c male mice were each administered the test article at
doses of 5 x 107 , 5 x 10 8, and 5 x 109 CPU/mouse both intravenously and
intraperitoneally, and orally at doses of 5 x 107, 5 x 10 8, and 2 x 10 11 CFU/mouse
(control group mice were given sterile PBS). Animals were observed for seven days,
and on days two and seven, five animals from each group were euthanized and
internal organs were observed macroscopically. For the groups treated orally,
tissues were collected for histopathological examination (liver, kidneys, lungs,
spleen, intestine, mesenteric lymph nodes, brain, thymus, and tissues around the
throat). There were no treatment related deaths, even in groups given the strain

intravenously. There were no adverse effects (AEs) observed related to activity and
weight. All animals were reported to be clinically healthy. There were no
differences in visceral organ appearance or histopathological examinations between
treated and control groups. The authors also described a 10-day repeated dose study
using oral administration in groups of ten mice (1 x 10 6 CFU/day) , rabbits (1 x 109
CFU/day), and piglets ( 1 x 10 9 CFU/day), as well as a 30-day repeated dose study
using groups of ten rabbits. There were no AEs noted or changes in hematology
values, or gross or histopathological findings compared to controls.
Hong et al. (2008) performed a repeated-dose gavage study of B. subtilis natto in 6
male New Zealand White rabbits as compared to an equal number of controls. 48 A
dose of 1 x 109 spores was given to the treated animals daily for 30 days. Blood
samples were taken on the last day and the liver, kidneys, spleen, small intestines,
and mesenteric lymph nodes were collected for histopathological examination.
There were no AEs in health status or feed intake, and no changes in hematology or
visceral organs or tissues were observed as compared to controls. The authors
additionally studied a single dose (1 x 10 12 CFU) of B. subtilis natto in guinea pigs,
as briefly described in the same publication. There were no findings related to
appetite, behavior, feces, weight gain, or histopathology 17 days after
administration in feed .
Tompkins et al. (2008) performed a 28-day repeated dose study in three groups of
ten Sprague-Dawley albino rats using 2 x 109 CFU/kg bw/day B. subtilis RO 179 or
E. faecium R0026 or control administered by gavage. 49 Animals were monitored
daily for potential signs of toxicity and groups were compared for mortality,
morbidity, behavior, body mass, food consumption, gross pathology, intestinal
colonization, and infection. Any changes in skin, fur, eyes, mucous membranes,
secretions/excretion, autonomic activity, gait, posture, handling response, sensory
reactivity, and movement were noted. At the end of treatment, the liver, kidneys,
spleen, heart, and lungs were subjected to histopathology and microbiological
exams. No findings, other than a lower heart mass (10%) in female rats, were noted.
The heart to body weight ratio was not affected by the treatment in these animals,
and no histopathological findings were mentioned. The B. subtilis strain was not
observed microbiologically except in the intestinal content of treated animals.
Additionally, Cell-Free Supematants (CFSs) of B . subtilis KATMIRA were
evaluated by a bacterial reverse mutation assay (Ames Salmonella assay) and
showed no mutagenicity. 50
6.3.2. Unpublished Acute Toxicity Study on B. subtilis Bss-19

An initial limit dose of 5000 mg/kg B. subtilis Bss-19 was administered to one
healthy female Sprague-Dawley rat by oral gavage. Due to the absence of mortality
in this animal, two additional female rats received the same dose. All animals were
observed for mortality, signs of gross toxicity, and behavioral changes at least once
daily for 14 days after dosing. Body weights were recorded prior to administration

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and again on days 7 and 14. Necropsies were performed on all animals at terminal
sacrifice. All animals survived test substance administration, gained body weight,
and appeared active and healthy during the study. There were no signs of gross
toxicity, AEs or abnormal behavior. No gross abnormalities were noted upon
necropsy. The LDso was determined to be greater than 5000 mg/kg bw in female
rats.
6.3.3. Human Studies

The safety of Bss-19 has not been formally investigated in healthy adult subjects.
However, many recent human clinical studies have been and continue to be
published on the strain of closest similarity, B. subtilis DEll l and on other B.
subtilis strains. 51 -54 Due to the large amount of published human studies, included
below are clinical studies located in the literature published since EFSA's most
recent Opinion in 2016, which addressed studies published up to June 2016.
6.3.3.1. B. subtilis DE111

In a human clinical trial investigating the safety of B. subtilis DEl 11 , Maher (2019)
gave 5 x 109 CFU B. subtilis DEl 11 in a single capsule or placebo daily to 41
healthy young adults for an average of 20 days. 55 Blood samples (comprehensive
metabolic panel, lipid panel, C-reactive protein (CRP)) and stool samples were
collected at the beginning and end of the study. Serum glucose levels were
significantly lower in the treatment group when comparing pre to post capsule
consumption. Triglycerides remained the same within the treatment group, while
the control group displayed a significant increase from pre to post capsule
consumption. There was no significant variation from the normal range of CRP. The
authors discussed that the decrease in serum glucose had been observed in hvo
animal studies where, in the first study, a compound isolated from B. subtilis (1-
Deoxynojirimycin) helped to improve diabetic conditions in bovine calves and, in a
second study, freeze-dried cultures of a combination of bacte1ia reduced blood
glucose levels in rats with elevated glucose levels. The authors concluded that daily
ingestion of one capsule containing approximately 5 x 109 CPU B. subtilis was well
tolerated in healthy young adults.
Cuentas et al. (2017) investigated the use of 1 x 109 CFU B. subtilis DE 111 or
placebo daily for 105 days in 50 adults. Comprehensive metabolic panels, lipid
panels, and CRP levels stayed within normal references ranges for the treatment and
placebo groups with no significant serum level differences. Additionally, no AEs
were reported. 56
In addition to the safety study and clinical study above, the following two clinical
studies were conducted which did not have any reported AEs. Toohey et al. (2018)
investigated the effects of 5 x 109 CFU B. subtilis DEl 11 or placebo daily on 23
Division I female athletes for 10 weeks-no AEs were reported. 57 Townsend et al.
(2018) gave 1 x 109 CPU B. subtilis DEl 11 or placebo daily to 25 Division I male
athletes for 12 weeks-no AEs were reported. 58

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6.3.3.2. Other B. Subtilis Strains

Penet et al. (2019) gave 5 x 109 CFU s of B. subtilis MB40 or placebo daily to 100
subjects for four weeks. 59 There were no statistically significant differences between
groups with regard to anthropometric, vital, hematological and clinical chemistry
parameters. Reported AEs were similar between groups and consisted of
constipation, diarrhea, flatulence, dry mouth, abdominal discomfort, increased
appetite and paresthesia. All AEs were resolved before end-of-study. Soman and
Swamy (20 19) evaluated the safety in a combination of 2 x 109 CFUs of B.
coagulans, B. clausii, and B. subtilis versus placebo in 60 subjects for 30 days. 60 No
AEs were reported in any subject during the study period. Hatanaka et al. (2018)
gave 2.2 X 109 B. subtilis C3102 spores or placebo daily to 88 healthy adults with
loose stool for eight weeks and did not rep01t any AEs. 51 Lefevre et al. (2017)
described that after 40 total days of treatment with B. subtilis CU 1 (2 x 109
spores/day) compared to placebo in 100 elderly human subjects, no undesirable
physiological effects or biological safety concerns with regard to AEs, liver and
kidney function markers, complete blood counts, hemodynamic parameters, and
vital signs were noted. 52 , 6 1 Alkaya et al. (2017) investigated the use of 5 x 107
CFU/day of combined B. subtilis, B. megaterium, and B. pumilus spores in subjects
for eight weeks- no AEs occurred. 53
Further, one study published before the most recent EFSA Opinion, Hanifi et al.
(2015), is included because it evaluated oral dose-response tolerance at similar or
higher daily dosages than the intended use levels of Bss-19. To evaluate oral dose
response tolerance, Hanifi et al. (2015) gave 81 subjects B. subtilis RO 179 at doses
of 0.1 x 109, 1 x 109, and 10 x 109 CFU/day or placebo for 4 weeks. The test article
was well tolerated at all doses and survived passage through the human GI tract. 54
6.3.4 Opportunistic Infections

Rare infections caused by B. subtilis have been described in the literature. For
example, a 73 year old male with chronic lymphocytic leukemia had a positively
identified recurrent septicemia caused by B. subtilis .62 Another case report in the
literature involved a patient with an esophageal perforation who had bacteremia and
mediastinitis due to co-infection with B. subtilis and B. licheniformis. 63 Overall,
infections with B. subtilis occur at very low rates, and generally occur in hospital
settings in immunocompromised patients and/or during medical procedures. 62 -64
6.4 Allergenicity
Bss-19 does not contain or have added any of the eight major allergens (milk, egg,
fish, Crustacean shellfish, tree nuts, wheat, peanuts, and soybeans) identified, and
required to be disclosed in labeling, in the Food Allergen Labeling and Consumer
Protection Act (F ALCPA). No reports of allergic reactions to B. subtilis were found
in our investigations.

,l AIBMR Life Sciences, Inc.
No reports of allergic reactions to B. subtilis were found in our investigations. Given

total exposure together with results of toxicological and clinical studies, the allergic
potential of B. subtilis can be considered very low.
6.5 Past Sales and Reported Adverse Events

Bss-19 has never been released on the market and therefore has no past sales or past
reported AEs to account for. No FDA letters regarding concern for safety to
companies that market products containing B. subtilis were located. A search of
FDA's Recalls, Market Withdrawals, & Safety Alerts search engine and FDA's
Center for Food
Safety and Applied Nutrition Adverse Event Reporting System did not uncover any
mention of B. subtilis products. All databases were accessed on June 2, 2020.
6.6 Basis for the GRAS Conclusion

Dupont's Bss-19 has been the subject of a thorough safety assessment as described
above. The totality of evidence supporting safety is comprised of data and
information that establish the safety of Bss-19 under the conditions of its intended
use and data and information that is corroborative of safety. The general availability
and general acceptance, throughout the scientific community of qualified experts,
of the data and information that establish the safety of Bss-19 under its intended
conditions of use establish the general recognition of this data and information.
Together, the establishment of safety based on scientific procedures and its general
recognition form the basis for DuPont's conclusion of GRAS status of Bss-19 for
its intended use.
6.6.1 Data and Information that Establish Safety

The scientific data, information, and methods forming the basis of this conclusion
are:
• The establishment of identity via l 6S rRNA sequence as well as complete
genome sequencing, demonstrating unequivocally that it is a strain of the B.
subtilis subspecies inaquosorum with established phenotypic characteristics;
• The analyses and resulting data showing Bss-19 lacks resistance to clinically
relevant antibiotics per European Food Safety Authority (EFSA) minimal
inhibitory concentration cut-offs and guidelines, with the exception of
chloramphenicol, where further investigation by DuPont showed that the
resistance is not expected to be transferrable;
• The lack of potential of Bss-19 to produce toxins or virulence factors that
have been demonstrated to be virulent to hosts (via comparison of genomic
sequences to known virulence sequences in the DBETH exotoxin protein
database);

. . . AlBMR Life Sciences, Inc.
• The methods of manufacture, specifications, as well as batch analyses,

showing that all specifications are met for each batch, demonstrating safe
production methods and robust quality control standards for Bss-19;
• The intended use as an ingredient in foods at an addition level of up to 1 x
°
10 1 CFU per serving, which is in line with addition levels for other GRAS
microbial ingredients (including B. subtilis in GRN 831) as well as with
levels of fermenting bacteria found naturally in various fermented foods,
with an estimated exposure of9.1 x 10 10-1.4 x 10 11 CFU/day (1.3-2.0 x 109
CFU/kg bw/day) by conservatively assuming consumption at the maximum
intended use addition level in 50% of all food servings daily;
• A previous GRAS notice to FDA (GRN 831) for a very similar strain, B.
subtilis inaquosorum strain DE 111, received a no questions letter from FDA
for use as an ingredient in cow's milk and soy-based non-exempt infant
formula for term infants at a maximum level of 2 x 108 CFU/100 mL and in
baked goods and baking mixes; beverages and beverage bases; breakfast
cereals; chewing gum; coffee and tea; condiments and relishes; confections
and frostings; dairy product analogs; fats and oils; fruit juices; frozen daily
deserts and mixes; fruit and water ices; gelatins; puddings and fillings; grain
products and pastas; soft/hard candy and cough drops; herbs, seeds, spices,
seasonings, blends, extracts, and flavorings; jams and jellies; milk and milk
products; nuts and nut products; plant protein products; processed fruits;
processed vegetables and vegetable juices; snack foods; soups and soup
mixes; sugar; and sweet sauces, toppings, and syrups at addition levels from
°
1 x 106 to 1 x 10 1 CFU/serving. The estimated daily intake (EDI) for the
strain was determined to be 1.3 x 10 11 CFU/day which FDA did not object
to.
• Another previous GRAS notice to FDA (GRN 905) for another B. subtilis
strain, B, subtilis DSM 32444, received a no questions letter from FDA for
use as in ingredient in beverages (milk drinks, protein high energy sports
drinks, hot beverages, and juices) and dry and shelf-stable products (cereals,
cookies, gums, and confectionary) at a maximum level of 1 x 109
CFU/serving. The EDI for the strain was determined to be 5.0 x 10 9 CFU/day
which FDA did not object to.
6.6.2 Data and Information that are Corroborative of Safety

• B. subtilis' EFSA QPS status for food and feed use, at any reasonable
dose/intended use, suggesting no further regulatory review prior to
introduction of new strains into the European food supply, other than the
qualifications that it must be verified to not possess toxigenic traits or harbor
acquired antimicrobial resistance genes;
• The documented long history of safe human consumption of B. subtilis as a
common bacterial species in fermented foods, 38 such as in natto (with

1"l AIBMR Life Sciences, Inc.
concentrations of approximately 1 x 10 8 CFU/gram, equivalent to

°
approximately 1 x 10 1 CFU/ 100 g serving), over decades without known
concerns for safety;48 • 49
• The lack of serious adverse events reported in clinical trials using B. subtilis
°
at daily dosages up to 1 x 10 1 CFU/day;
• Agreement in the literature that it is highly unlikely that a microorganism
maintained in pure culture, with a histo1y of safe use, would become unsafe
as a result of mutation (genetic drift), production changes, or delivery format
changes; 65 -67
• An unpublished acute oral toxicity study showing the acute oral LDso of B.
subtilis Bss-19 is greater than 5000 mg/kg bw in female rats.
6.6.3 General Recognition

The scientific data, information, and methods herein reported, that provide the basis
of this GRAS conclusion by scientific procedures are published and available in the
public domain. Part 7 of this GRAS notice contains the citations for the published
studies. These publicly available data and information fulfill the requirement of the
GRAS standard for general availability of the scientific data, information, and
methods relied on to establish the safety ofBss- 19 for its intended conditions of use.
The peer-review of the published studies and lack of Letters to the Editor or other
dissenting opinions provide ample evidence of general recognition among qualified
experts that there is reasonable certainty that consumption of Bss- 19 for its intended
use is not harmful. The general availability and acceptance of these scientific data,
information, and methods satisfy the criterion of the GRAS standard that general
recognition of safety requires common knowledge throughout the scientific
community knowledgeable about the safety of substances directly or indirectly
added to food that there is reasonable certainty that the substance is not harmful
under the conditions of its intended use.
6.6.4 Data and Information that are Inconsistent with the GRAS Conclusion
We have reviewed the available data and information and are not aware of any data
and information that are, or may appear to be, inconsistent with our conclusion of
GRAS status.
6.6.5 Information that is Exempt from Disclosure under FOIA

There are no data or information in this report that are considered trade secret or
commercial or financial information that is privileged or confidential.

Part 7: Supporting Data and Information

Literature searches for the safety assessment described in Part 6 of this GRAS notice
were conducted through July 2019 and again on June 2, 2020.
7.1 Data and Information that are not Generally Available

An unpublished acute oral toxicity study was provided by DuPont and is part of the
basis of the determination of safety (subpart 6.3.2).
7.2 References that are Generally Available
1. Kunst F, Ogasawara N, et al. The complete genome sequence of the gram

positive bacterium Bacillus subtilis. Nature. 1997;390(6657):249-56
2. Permpoonpattana P, Hong HA, et al. Evaluation of Bacillus subtilis strains
as probiotics and their potential as a food ingredient. Bene/ Microbes.
2012;3(2): 127-35
3. EFSA. Introduction of a Qualified Presumption of Safety (QPS) approach
for assessment of selected microorganisms referred to EFSA. Opinion of the
Scientific Committee. The EFSA Journal. 2007;587:1-16
4. Darling AC, Mau B, et al. Mauve: multiple alignment of conserved genomic
sequence with rearrangements. Genome Res. 2004;14(7):1394-403
5. EFSA. Scientific Opinion. Guidance on the assessment of bacterial
susceptibility to antimicrobials of human and veterinary importance. EFSA
Journal. 2012;10(6):2740
6. Ammor MS, Florez AB, et al. Antibiotic resistance in non-enterococcal lactic
acid bacteria and bifidobacteria. Food Microbial. 2007;24(6):559-70
7. Balcazar JL. Bacteriophages as vehicles for antibiotic resistance genes in the
environment. PLoS Pathog. 2014;10(7):e1004219
8. Bennett PM. Plasmid encoded antibiotic resistance: acquisition and transfer
of antibiotic resistance genes in bacteria. Br J Pharmacol. 2008;153 Suppl
l:S347-57
9. Mathur S and Singh R. Antibiotic resistance in food lactic acid bacteria--a
review. Int J Food Microbial. 2005;105(3):281-95
10. WHO. The world is running out of antibiotics, WHO report confirms; 2017.
11. FAO/WHO. Guidelines for the evaluation of probiotics in food. 2002.1-11.
12. EFSA Panel on Biological Hazards (BIOHAZ), Ricci A, et al. Scientific
Opinion on the update of the list of QPS-recommended biological agents
intentionally added to food or fee d as notified to EFSA. EFSA Journal.
2017;15(3):177
13. Bernardeau M, Vernoux JP, et al. Safety assessment of dairy
microorganisms: the Lactobacillus genus. Int J Food Microbial.
2008;126(3):278-85
14. EFSA Panel on Additives and Products or Substances used in Animal Feed
(FEEDAP). Guidance on the characterisation of microorganisms used as
feed additives or as production organisms. EFSA Journal. 2018;16(3):5206

,~I\
15.
Jia B, Raphenya AR, et al. CARD 2017: expansion and model-centric

curation of the comprehensive antibiotic resistance database. Nucleic Acids
Res. 2017;45(Dl):D566-D573
16. Siguier P, Perochon J, et al. ISfinder: the reference centre for bacterial
insertion sequences. Nucleic Acids Res. 2006;34(Database issue):D32-6
17. EFSA Panel on Additives and Products or Substances used in Animal Feed
(FEEDAP). Scientific Opinion on the safety and efficacy of Oralin®
(Enterococcus faecium) as a feed additive for calves for rearing, piglets,
chickens for fattening, turkeys for fattening and dogs. EFSA Journal.
2014;12(6):3727
18. Chakraborty A, Ghosh S, et al. DBETH: a Database of Bacterial Exotoxins
for Human. Nucleic Acids Res. 2012;40(Database issue):D615-20
19. Chen L, Yang J, et al. VFDB: a reference database for bacterial virulence
factors. Nucleic Acids Res. 2005;33(Database issue):D325-8
20. Spano G, Russo P, et al. Biogenic amines in fermented foods. Eur J Clin Nutr.
2010;64 Suppl 3:S95-100
21. EFSA Panel on Biological Hazards (BIOHAZ). Scientific Opinion on risk
based control of biogenic amine formation in fermented foods. EFSA
Journal. 2011;9(109):2393
22. Stiles ME and Holzapfel WH. Lactic acid bacteria of foods and their current
taxonomy. Int J Food Microbial. 1997;36(1):1-29
23. Ewaschuk JB, Naylor JM, et al. D-lactate in human and ruminant
metabolism. J Nutr. 2005;135(7):1619-25
24. Petersen C. D-lactic acidosis. Nutr Clin Pract. 2005;20(6):634-45
25. Connolly E, Abrahamsson T, et al. Safety of D(- )-lactic acid producing
bacteria in the human infant. J Pediatr Gastroenterol Nutr. 2005;41(4):489-92
26. Haschke-Becher E, Brunser 0, et al. Urinary D-lactate excretion in infants
receiving Lactobacillus johnsonii with formula. Ann Nutr Metab. 2008;53(3-
4):240-4
27. Haschke-Becher E, Baumgartner M, et al. Assay of D-lactate in urine of
infants and children with reference values taking into account data below
detection limit. Clin Chim Acta. 2000;298(1-2):99-109
28. Hove H and Mortensen PB. Colonic lactate metabolism and D-lactic
acidosis. Dig Dis Sci. 1995;40(2):320-30
29. Uribarri J, Oh MS, et al. D-lactic acidosis. A review of clinical presentation,
biochemical features, and pathophysiologic mechanisms. Medicine
(Baltimore). 1998;77(2):73-82
30. Munakata S, Arakawa C, et al. A case of D-lactic acid encephalopathy
associated with use of probiotics. Brain Dev. 2010;32(8):691-4
31. Ku W, Lau D, et al. Probiotics Provoked D-lactic Acidosis in Short Bowel
Syndrome: Case Report and Literature Review. HK J Paediatr. 2006;11:246-
254
32. de Vrese M, Koppenhoefer B, et al. D-lactic acid metabolism after an oral
load of DL-lactate. Clin Nutr. 1990;9(1):23-8
33. Rezac S, Kok CR, et al. Fermented Foods as a Dietary Source of Live
Organisms. Front Microbial. 2018;9:1785
34. Basiotis P, Lino M, et al. Consumption of Food Group Servings: People's
Perceptions vs. Reality. Nutrition Insights. 2000. 20: 1-2

~ AlBMR Life Sciences, Inc.
35. Millen AE, Midthune D, et al. The National Cancer Institute diet history
questionnaire: validation of pyramid food servings. Am J Epidemiol.
2006;163(3):279-88
36. Hong HA, Due le H, et al. The use of bacterial spore formers as probiotics.
FEMS Microbial Rev. 2005;29(4):813-35
37. Shimizu T. Health claims on functional foods: the Japanese regulations and
an international comparison. Nutr Res Rev. 2003;16(2):241-52
38. International Dairy Federation. Safety demonstration of microbial food
cultures (MFC) in fermented food products. Bulletin of the International
Dairy Federation 455 / 2012. 2012. 62.
39. International Dairy Federation. Inventory of microbial food cultures with
safety demonstration in fermented food products. Update of the Bulletin of
the IDF No. 455/2012: Safety demonstration of Microbial Food Cultures
(MFC) in fermented food cultures. Bulletin of the International Dairy
Federation 495/2018. 2018. 71.
40. EFSA Panel on Biological Hazards (BIOHAZ). Update of the list of QPS
recommended biological agents intentionally added to food or feed as
notified to EFSA 10: Suitability of taxonomic units notified to EFSA until
March 2019. EFSA Journal . 2019;17(7):5753
41. EFSA Panel on Biological Hazards (BIOHAZ). Update of the list of QPS
recommended biological agents intentionally added to food or feed as
notified to EFSA 5: suitability of taxonomic units notified to EFSA until
September 2016. EFSA Journal. 2016;15(3)
42. Aguilera J, Gomes AR, et al. Principles for the risk assessment of genetically
modified microorganisms and their food products in the European Union.
Int J Food Microbial. 2013;167(1):2-7
43. Biopesticides and Pollution Prevention Division, Office of Pesticide
Programs, et al. Biopesticide Registration Action Document. Bacillus
subtilis strain QST 713. 2006. 41.
44. Zhang C, Zhang Y, et al. Toxicological and safety evaluation of Bacillus
subtilis strain Tpb55. Advanced Materials Research. 2013;807-809:1947-1953
45. Kim K-H, Jeong C, et al. Single dose oral toxicity of Bacillus subtilis JNS in
ICR mice [Abstract only]. J Korean Soc Food Sci Nutr. 2015;44(1):24-28
46. Nakamura H, Imazawa T, et al. A 90-day subchronic oral toxicity study of
Bacillus subtilis gum in F344 rats [In Japanese. English abstract only] .
Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku . 1999(117):119-22
47. Sorokulova IB, Pinchuk IV, et al. The safety of two Bacillus probiotic strains
for human use. Dig Dis Sci. 2008;53(4):954-63
48. Hong HA, Huang JM, et al. The safety of Bacillus subtilis and Bacillus
indicus as food probiotics. JAppl Microbial. 2008;105(2):510-20
49. Tompkins TA, Hagen KE, et al. Safety evaluation of two bacterial strains
used in Asian probiotic products. Can J Microbial. 2008;54(5):391-400
50. AlGburi A, Volski A, et al. Safety properties and probiotic potential of
Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895.
Advances in Microbiology. 2016;6(6):432-452
51. Hatanaka M, Yamamoto K, et al. Effect of Bacillus subtilis C-3102 on loose
stools in healthy volunteers. Benef Microbes. 2018;9(3):357-365

52. Lefevre M, Racedo SM, et al. Safety assessment of Bacillus subtilis CUl for
use as a probiotic in humans. Regul Toxicol Pharmacol. 2017;83:54-65
53. Alkaya B, Laleman I, et al. Clinical effects of probiotics containing Bacillus
species on gingivitis: a pilot randomized controlled trial. J Periodontal Res.
2017;52(3 ):497-504
54. Hanifi A, Culpepper T, et al. Evaluation of Bacillus subtilis R0179 on
gastrointestinal viability and general wellness: a randomised, double-blind,
placebo-controlled trial in healthy adults. Benef Microbes. 2015;6(1):19-27
55. Maher M. Tolerance and effect of a probiotic supplement delivered in
capsule form. Food and Nutrition Sciences. 2019;10(6):626-634
56. Cuentas A, Deaton J, et al. The effect of Bacillus subtilis DEl 11 on the daily
bowel movement profile for people with occasional gastrointestinal
irregularity. J Prob Health. 2017;5(4):10000189
57. Toohey JC, Townsend JR, et al. Effects of Probiotic (Bacillus subtilis)
Supplementation During Offseason Resistance Training in Female Division
I Athletes. J Strength Cond Res. 2018
58. Townsend JR, Bender D, et al. Effects of Probiotic (Bacillus subtilis DE111)
Supplementation on Immune Function, Hormonal Status, and Physical
Perforn;,.ance in Division I Baseball Players. Sports (Basel) . 2018;6(3)
59. Penet C, Kramer R, et al. A Randomized, Double-blind, Placebo-controlled,
Parallel Study Evaluating the Efficacy of Bacillus subtilis MB40 to Reduce
Abdominal Discomfort, Gas, and Bloating. Altern Ther Health Med. 2019
60. Soman RJ and Swamy MV. A prospective, randomized, double-blind,
placebo-controlled, parallel-group study to evaluate the efficacy and safety
of SNZ TriBac, a three-strain Bacillus probiotic blend for undiagnosed
gastrointestinal discomfort. Int J Colorectal Dis. 2019;34(11):1971-1978
61. Lefevre M, Racedo SM, et al. Probiotic strain Bacillus subtilis CUl
stimulates immune system of elderly during common infectious disease
period: a randomized, double-blind placebo-controlled study. Immun
Ageing. 2015;12:24
62. Oggioni MR, Pozzi G, et al. Recurrent septicemia in an
immunocompromised patient due to probiotic strains of Bacillus subtilis. J
Clin Microbial. 1998;36(1):325-6
63. Jeon YL, Yang JJ, et al. Combined Bacillus licheniformis and Bacillus subtilis
infection in a patient with oesophageal perforation. J Med Microbial.
2012;61(Pt 12):1766-9
64. Matsumoto S, Suenaga H, et al. Management of suspected nosocomial
infection: an audit of 19 hospitalized patients with septicemia caused by
Bacillus species. Jpn J Infect Dis. 2000;53(5):196-202
65. Pariza MW, Gillies KO, et al. Determining the safety of microbial cultures
for consumption by humans and animals. Regul Toxicol Pharmacol.
2015;73(1):164-71
66. Stevens Hand Nabors L. Microbial food cultures: a regulatory update. Food
Tech. 2009;63(3):36-41
67. Sanders ME, Klaenhammer TR, et al. Effects of genetic, processing, or
product formulation changes on efficacy and safety of probiotics. Ann N Y
Acad Sci. 2014;1309:1-18

Viebrock, Lauren
From: Amy Clewell <amy@aibmr.com>

Sent: Friday, May 7, 2021 5:01 PM
To: Viebrock, Lauren
Cc: John Endres; Jared Brodin
Subject: [EXTERNAL] Re: DUPONT: GRN 000969 Questions
Attachments: Notifier Final Responses to 2021_04_30 GRN 969 Questions May 7 2021.pdf
Follow Up Flag: Follow up

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CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the
sender and know the content is safe.

Dear Dr. Viebrock,

Please find attached the notifier’s responses to the GRN 969 questions. Please don’t hesitate to let me know if any other
questions come up or if you need any clarification on the attached. Wishing you a nice weekend!
Kind Regards,
Amy Clewell, ND, DABT

Vice President, Scientific and Regulatory Affairs
(253) 286-2888
www.aibmr.com | @AIBMRInc
The information contained in this transmission may be legally privileged and confidential information intended only for the
use of the intended recipient. If you are not the intended recipient, the review, dissemination, distribution, copying, or printing
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From: "Viebrock, Lauren" <Lauren.Viebrock@fda.hhs.gov>
Date: Friday, April 30, 2021 at 10:42 AM
To: "jessica@aibmr.com" <jessica@aibmr.com>
Subject: GRN 000969 Questions

Dear Ms. Gruber,

During our review of GRAS Notice No. 000969, we noted questions that need to be addressed. Please find the questions
attached to this email.

We respectfully request a response within 10 business days. If you are unable to complete the response within that
time frame, please contact me to discuss further options.

If you have questions or need further clarification, please feel free to contact me. Thank you in advance for your
attention to our comments.

1
Regards,
Lauren

Lauren VieBrock
Regulatory Review Scientist/Microbiology Reviewer
Office of Food Additive Safety
U.S. Food and Drug Administration

Tel: 301-796-7454
lauren.viebrock@fda.hhs.gov
U.19 FOOD & DRUI

ADM JNIITH11 ON
D CJ C .. 11

2
,l
A IBM R
Life Sciences. Inc.
Lauren VieBrock
Tel: 301-796-7454
May 7, 2021
Re: Responses to FDA’s GRN 969 Questions
Dear Dr. VieBrock,

Please find responses to FDA’s questions concerning Bacillus subtilis Bss-19 (GRN 969) below. FDA’s
questions are in BLACK, while the notifier responses are in BLUE:
1. Danisco states that AOAC method 984.27 was used for the determination of arsenic, cadmium and
lead content. AOAC method 984.27 is validated for the determination of calcium, copper, iron,
magnesium, manganese, phosphorus, potassium, sodium, and zinc in infant formula using ICP-OES.
Please clarify what method was used to provide the results in the Table 3 for As, Cd and Pb. The actual
method used for As, Cd, and Pb determination in Bacillus subtilis Bss-19 is AOAC 2013.06.
2. Please provide the specific method for the analysis of mold/yeast in the cited USP41-NF36
compendium. The actual method used for mold/yeast analysis is FDA BAM Chapter 18. Please see the
revised specification table for Bacillus subtilis Bss-19 below:
Tested Parameters Specification Method

Viable cell count (CFU/g) ≥ 2.25 x 1011 In-house method
Color1 White to cream Visual Inspection
Form1 Freeze-dried Visual Inspection
powder
Heavy Metals2
Arsenic (ppm) < 1.0 ppm AOAC 2013.06
Cadmium (ppm) < 0.2 ppm AOAC 2013.06
Mercury (ppm) < 0.05 ppm AOAC 2013.06
Lead (ppm) < 0.5 ppm AOAC 2013.06
1425 Broadway
Suite 458
Seattle, WA 98122
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www.aibmr.com
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,l
A IBM R
Life Sciences. Inc.
Microbiological Tests
Coliform (CFU/g) < 10 AOAC 991.14
Escherichia coli (CFU/g) < 10 AOAC 991.14
Salmonella Negative in 50 g AOAC RI-121501
Listeria Negative in 25 g FDA BAM Chapter 10
Staphylococcus coag+ (CFU/g) < 10 FDA BAM Chapter 12
B. cereus (CFU/g) < 10 AOAC 980.31
Molds and Yeast3 < 100 CFU/g FDA BAM Chapter 18
Abbreviations: CFU, colony forming unit; AOAC, Association of Official Analytical Chemists; FDA BAM, Food and Drug
Administration’s Bacteriological Analytical Manual; ppm, parts per million
1
Internal Specification, not reported on CoA
2
Based on annual surveillance testing, not generally reported on CoA
3
Tested on bulk intermediate powder, not generally reported on CoA
3. At the time of the submission of the notice, Danisco stated that stability studies are underway. Please
provide results from the stability studies. The results of 24 months of stability testing for Bacillus
subtilis Bss-19 under three different testing conditions (temperature and relative humidity) are shown
below:
Results in Testing
Months
CFU/g Condition
loss
0 1 3 6 9 12 18 24 after 24
months
Batch 1 25°C
4.30E+11 4.10E+11 5.20E+11 4.60E+11 4.53E+11 5.73E+11 5.30E+11 4.03E+11 6%
(1493413902) 60% RH
Batch 1 30°C
4.30E+11 4.43E+11 5.03E+11 3.53E+11 5.07E+11 4.10E+11 4.30E+11 2.80E+11 35%
(1493413902) 65% RH
Batch 1 40°C
4.30E+11 4.70E+11 4.60E+11 5.40E+11 2.97E+11 2.70E+11 1.53E+11 7.83E+10 82%
(1493413902) 75% RH
Batch 2 25°C
4.30E+11 5.77E+11 4.40E+11 4.90E+11 5.93E+11 5.10E+11 4.70E+11 5.37E+11 -25%
(1493438028) 60% RH
Batch 2 30°C
4.30E+11 4.33E+11 5.10E+11 4.87E+11 4.57E+11 4.27E+11 5.57E+11 2.57E+11 40%
(1493438028) 65% RH
Batch 2 40°C
4.30E+11 4.70E+11 3.87E+11 4.33E+11 2.23E+11 1.36E+11 1.12E+11 7.27E+10 83%
(1493438028) 75% RH
Batch 3 25°C
4.03E+11 4.77E+11 3.90E+11 3.27E+11 3.23E+11 2.93E+11 3.40E+11 3.40E+11 16%
(1493441558) 60% RH
Batch 3 30°C
4.03E+11 4.67E+11 3.80E+11 3.77E+11 3.83E+11 3.73E+11 2.80E+11 2.19E+11 46%
(1493441558) 65% RH
Batch 3 40°C
4.03E+11 4.90E+11 4.27E+11 3.20E+11 1.84E+11 1.31E+11 4.07E+10 4.93E+10 88%
(1493441558) 75% RH
1425 Broadway
Suite 458
Seattle, WA 98122
(253) 286-2888 ph
www.aibmr.com
www.toxicoop.com 2
1l
A IBM R
Life Sciences. Inc.
Overall, the average loss in the three batches by condition over 24 months are as follows:
• 25°C/60% RH = -1%
• 30°C/65% RH = 40%
• 40°C/75% RH = 84%
4. Please clarify whether the strain has been deposited in a recognized culture collection. The strain has
been deposited in the American Type Culture Collection (ATCC) with an ID of “SD-7780”. ATCC
confirmed receipt of the strain on May 28th, 2020.
5. Please provide more information on the proportions of spores to vegetative cells in the spore
preparation. At the end of the manufacturing process, the spray dried bacteria are heat shocked to kill all
non-spore bacteria (~80 °C for 15 minutes). Thus, the final ingredient consists of essentially 100%
spores.
6. Please clarify whether or not the intended use in beverages includes intended use in alcoholic
beverages. The notifier would like to include alcoholic beverages as an intended use for Bacillus subtilis
Bss-19. Please let us know if you have any concerns with this use of the ingredient.
Sincerely,

Vice Presendent, Scientific and Regulatory Affairs
AIBMR Life Sciences, Inc., on behalf of Danisco USA, Inc.
1425 Broadway
Suite 458
Seattle, WA 98122
(253) 286-2888 ph
www.aibmr.com
www.toxicoop.com 3
Viebrock, Lauren

Sent: Wednesday, September 1, 2021 5:05 PM
Cc: John Endres; Jared Brodin
Subject: Re: [EXTERNAL] Re: DUPONT: GRN 000969 Questions
Attachments: Notifier Final Responses to 2021_04_30 GRN 969 Questions Sept 1 2021.pdf

Dear Dr. Viebrock,
I hope that this email finds you well! Please find attached responses to FDA’s most recent questions related to GRN 969.
Please also don’t hesitate to let us know if you need any additional clarifications. If you could acknowledge that this was
received, we’d greatly appreciate it. Thank you!

Kind Regards,

(253) 286-2888

Date: Thursday, August 19, 2021 at 6:47 AM
To: Amy Clewell <amy@aibmr.com>
Cc: John Endres <john@aibmr.com>, Jared Brodin <Jared@aibmr.com>
Subject: RE: [EXTERNAL] Re: DUPONT: GRN 000969 Questions

Dear Dr. Clewell,

During our review of GRAS Notice No. 000969, we noted an additional question that need to be addressed.

‐On page 23 of the notice, the notifier states that uses in other foods (in addition to these listed in GRN 000831
and GRN 000905) may be considered more novel and additive with regards to exposure. FDA requests a
clarification what is meant by novel and additional uses. Please specify these additional use categories.



1

Regards,
Lauren

Lauren VieBrock


Tel: 301-796-7454
U.S. FOOD
-IDlll llltl-'11011
DRUI
D Cl l:l ··

Sent: Wednesday, May 12, 2021 12:30 PM
To: Viebrock, Lauren <Lauren.Viebrock@fda.hhs.gov>
Cc: John Endres <john@aibmr.com>; Jared Brodin <Jared@aibmr.com>


Thank you Dr. Viebrock!

Kind Regards,

(253) 286-2888

Date: Tuesday, May 11, 2021 at 7:58 AM

Dear Dr. Clewell,

2
Thank you for your responses to our questions regarding GRN 969. This is confirm receipt of the amendment submitted
on May 7, 2021.

Regards,
Lauren

Sent: Monday, May 10, 2021 10:42 PM


Dear Dr. Viebrock,
I am writing just to make sure that the responses to GRN 969 were received per the email below. Thank you in advance
for confirming!

Kind Regards,

(253) 286-2888


Date: Friday, May 7, 2021 at 2:00 PM
To: "Viebrock, Lauren" <Lauren.Viebrock@fda.hhs.gov>
Subject: Re: DUPONT: GRN 000969 Questions

Dear Dr. Viebrock,


Kind Regards,

(253) 286-2888

3


Dear Ms. Gruber,




Regards,
Lauren

Lauren VieBrock
Tel: 301-796-7454

!',;!;.~~!: DRUI
D CJ IZI ··

4
,l
A IBM R
Life Sciences. Inc.
Lauren VieBrock
Tel: 301-796-7454
September 1, 2021
Re: Responses to FDA’s GRN 969 Questions
Dear Dr. VieBrock,

Please find responses to FDA’s questions concerning Bacillus subtilis Bss-19 (GRN 969) below. FDA’s
questions are in BLACK, while the notifier responses are in BLUE:
1) On page 23 of the notice, the notifier states that uses in other foods (in addition to these listed in GRN
000831 and GRN 000905) may be considered more novel and additive with regards to exposure. FDA
requests a clarification what is meant by novel and additional uses. Please specify these additional use
categories. As you are aware, the intended use of B. subtilis Bss-19 in GRN 969 is as an ingredient
added to foods at a level of up to 1 x 1010 CFU/serving where standards of identity do not preclude such
use (note that this use does not including alcoholic beverages—see question 2 below. The GRN also
points out that it does not include use in infant formula or in foods that would require additional USDA
review). Some general examples were given on page 23 of the notice (“yogurt and other dairy products,
soy products, beverages, chewing gum, confectionary, snacks, and other foods”), however the examples
were not intended to be all-inclusive.
As noted, the GRN also states that “The addition of Bss-19 to some foods may be substitutive with
regard to other GRAS B. subtilis strains’ intended uses (e.g. GRN 831 and 905), or with regard to
traditional uses of B. subtilis. However, uses in other foods may be considered more novel and additive
with regard to exposure.” This statement was only meant to conservatively point out that while there is a
lot of overlap in the intended uses in GRN 969 compared to those in GRNs 831 and 905 as well as
traditional uses, there may hypothetically be uses that do not overlap and thus could be considered
“additional” (additive) or “novel” versus substitutive between B. subtilis strains. To clarify this further,
the intended uses stated in GRNs 831 and 905 are reviewed below:
1425 Broadway
Suite 458
Seattle, WA 98122
(253) 286-2888 ph
www.aibmr.com
www.toxicoop.com 1
I~
A IBM R
Life Sciences, Inc.
• GRN 831 states a large number of uses for its B. subtilis strain, but the list appears to
be finite (i.e. the list was not just examples, but instead was a full list of uses). The
list includes use in baked goods and baking mixes; beverages and beverage bases;
breakfast cereals; chewing gum; coffee and tea; condiments and relishes;
confections and frostings; dairy product analogs; fats and oils; fruit juices; frozen
dairy desserts and mixes; fruit and water ices; gelatins, puddings, and fillings; grain
products and pastas; soft/hard candy and cough drops; herbs, seeds, spices,
seasonings, blends, extracts, and flavorings; jams and jellies; milk and milk products;
nuts and nut products; plant protein products; processed fruits; processed
vegetables and vegetable juices; snack foods; soups and soup mixes; sugar; and
sweet sauces, toppings, and syrups at levels from 1 x 106 to 1 x 1010 CFU/serving.
• GRN 905 states that its B. subtilis strain is intended to be used as an “ingredient in
conventional foods at a level of 109 CFU/serving”.
Thus, while Bss-19 is a unique strain of B. subtilis, the uses of this strain could be substitutive for many
of the B. subtilis traditional or intended uses from other GRN strains. B. subtilis is generally used in
some traditional fermented foods as described in GRN 969, and thus use of the Bss-19 strain in such
foods may be considered substitutive for use of other B. subtilis strains if used in a similar manner. Use
of Bss-19 may also be substitutive in various foods with regard to the B. subtilis strain from GRN 831
since the addition levels are identical (up to 1010 CFU/serving) and because GRN 831 lists a large
number of intended use food categories. However, because Bss-19 is intended for use in any food
(except those categories stipulated above), there may be hypothetical instances where it is added to a
food not included in the GRN 831 list.
Similarly, while the B. subtilis strain in GRN 905 is intended for use in any conventional food, the
maximum addition level is slightly lower than the addition level stated for Bss-19 in GRN 969. Thus,
while use of the Bss-19 strain in conventional food categories would likely be substitutive to those of the
strain in GRN 905, Bss-19 may be added to some foods at a slightly higher addition level per serving
than that of the strain in GRN 905 (1010 versus 109 CFU per serving).
In summary, any hypothetical uses or use levels outside of those covered in the two GRNs listed above
or by traditional uses could be considered “additional” or “novel”. However, the notifier does not have
any specific “additional” or “novel” uses in mind at this time. We hope this makes sense, and please do
not hesitate to let us know if you need any additional clarification.
1425 Broadway
Suite 458
Seattle, WA 98122
(253) 286-2888 ph
www.aibmr.com
www.toxicoop.com 2
1l
A IBM R
Life Sciences. Inc.
2). Please clarify whether or not the intended use in beverages includes intended use in alcoholic
beverages. The notifier would like to withdraw “alcoholic beverages” as an intended use for Bacillus
subtilis Bss-19.
Sincerely,

Vice Presendent, Scientific and Regulatory Affairs
AIBMR Life Sciences, Inc., on behalf of Danisco USA, Inc.
1425 Broadway
Suite 458
Seattle, WA 98122
(253) 286-2888 ph
www.aibmr.com
www.toxicoop.com 3
Viebrock, Lauren

Sent: Monday, September 27, 2021 9:36 AM
Cc: John Endres; Jared Brodin; West-Barnette, Shayla

Hi Lauren and Shayla,

We communicated with the notifier regarding this question. The Bss‐19 ingredient is only spray‐dried. We apologize for
the mention of freeze‐drying in the submission. Dupont has other strains that are freeze‐dried and this appears to be a
typo related to that. Any mention of freeze‐drying should be replaced with spray‐draying.

Please let us know if you have any other questions or need any other information in this regard. Thank you!

Kind Regards,

(253) 286-2888

Date: Friday, September 24, 2021 at 6:18 PM
Cc: John Endres <john@aibmr.com>, Jared Brodin <Jared@aibmr.com>, "West‐Barnette, Shayla"
<Shayla.WestBarnette@fda.hhs.gov>

Hi Amy,

Thank you for the update. We look forward to receiving the response.

In the event that I am on leave when your response is submitted, please also include my colleague Dr. Shayla West‐
Barnette (whose contact information is copied on this email) and she will then become the Regulatory Review Scientist
and point of contact for your submission moving forward.

Best,
Lauren
1

Sent: Thursday, September 23, 2021 3:55 PM


Hi Lauren,
I just wanted to let you know that this question was received and I’m seeking feedback from Dupont about an answer.
I’ll send it to you as soon as we receive it.
Thanks!

Kind Regards,

(253) 286-2888

Date: Wednesday, September 22, 2021 at 6:26 AM

Hi Amy,

Thank you for confirming the notifier name.

One additional question on GRN 969. The ingredient description states it is a freeze‐dried powder, while the
manufacturing section describes the ingredient as spray‐dried. Can you please clarify whether the ingredient is spray‐ or
freeze‐dried. Thank you.

Best,
Lauren

Sent: Monday, September 13, 2021 9:53 PM

2

Hello again Lauren,
Our main contact for the project (i.e. the Notifier) responded as follows:
“Danisco USA, Inc. is still our legal entity, I think it would make most sense to have FDA continue to address letters to
this company name.”
Thanks very much for checking in on this, and let us know if there is anything else that you need!

Kind Regards,

(253) 286-2888

Date: Monday, September 13, 2021 at 8:56 AM

Good morning Lauren,
This is a great question! I will check with the client (notifier) and get back to you ASAP.

Kind Regards,

(253) 286-2888

Date: Monday, September 13, 2021 at 7:59 AM
3

Hi Amy,

Thank you for your response. The notifier is written as “Danisco USA, Inc” in the submission. With the recent
organizational changes at the company, is that how the notifier should still be addressed in our correspondences?

Thanks,
Lauren

Sent: Wednesday, September 1, 2021 5:05 PM


Dear Dr. Viebrock,
I hope that this email finds you well! Please find attached responses to FDA’s most recent questions related to GRN 969.
Please also don’t hesitate to let us know if you need any additional clarifications. If you could acknowledge that this was
received, we’d greatly appreciate it. Thank you!

Kind Regards,

(253) 286-2888

Date: Thursday, August 19, 2021 at 6:47 AM

Dear Dr. Clewell,

During our review of GRAS Notice No. 000969, we noted an additional question that need to be addressed.

‐On page 23 of the notice, the notifier states that uses in other foods (in addition to these listed in GRN 000831
and GRN 000905) may be considered more novel and additive with regards to exposure. FDA requests a
clarification what is meant by novel and additional uses. Please specify these additional use categories.

4


Regards,
Lauren

Lauren VieBrock


Tel: 301-796-7454
U.S. FOOD DRUB

4 PIii i ii 'i.ii 11011
D Cl m ,.

Sent: Wednesday, May 12, 2021 12:30 PM


Thank you Dr. Viebrock!

Kind Regards,

(253) 286-2888

Date: Tuesday, May 11, 2021 at 7:58 AM
5

Dear Dr. Clewell,

Thank you for your responses to our questions regarding GRN 969. This is confirm receipt of the amendment submitted
on May 7, 2021.

Regards,
Lauren

Sent: Monday, May 10, 2021 10:42 PM


Dear Dr. Viebrock,
I am writing just to make sure that the responses to GRN 969 were received per the email below. Thank you in advance
for confirming!

Kind Regards,

(253) 286-2888


Date: Friday, May 7, 2021 at 2:00 PM
Subject: Re: DUPONT: GRN 000969 Questions

Dear Dr. Viebrock,


Kind Regards,
6
(253) 286-2888



Dear Ms. Gruber,




Regards,
Lauren

Lauren VieBrock

Tel: 301-796-7454
llL FOOD & DRUI

o1 DIii I SI 1-111011
D Cl Etl ··

7
From: Victoria
Cc: West-Barnette, Shayla
Subject: RE: [EXTERNAL] RE: GRN 957
Date: Wednesday, October 6, 2021 9:07:04 AM
Attachments: image001.png
CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you
recognize the sender and know the content is safe.
Lauren, the 3mg level that manufacture uses is their maximum level so anything under that would
be detected. They have told me that they will work according the FDA U.S. metal limits and change
the upper limit for lead to 0.5 mg/kg.
Thank you
Victoria Davis
Prozure
(561)926.2723
This email (and any attachments) is only intended for the use of the person or entity to which it is
addressed, and may contain information that is privileged and confidential. You, the recipient, are
obligated to maintain it in a safe, secure and confidential manner. Unauthorized disclosure or failure to
maintain confidentiality may subject you to federal and state penalties. If you are not the intended
recipient, please immediately notify us by return email, and delete this message from your computer.
From: Viebrock, Lauren [mailto:Lauren.Viebrock@fda.hhs.gov]

Sent: Tuesday, September 28, 2021 3:22 PM
To: Victoria
Subject: RE: [EXTERNAL] RE: GRN 957
Hi Victoria,
Please find our additional question for GRN 957 below.
In response to questions raised during our review of your notice, you confirmed that there was no
expectation that heavy metal contaminants, including lead, are present or detectable in your
Lactobacillus johnsonii ingredient. The specified limit for lead presented in your GRAS notice is up to
3 mg/kg and the results of batch analyses were reported in certificates of analysis as <3 mg/kg, zero,
or ‘none detected.’ Although specification limits for contaminants are typically set a level higher
than the limits of the methods used for their detection, we note that the limit of 3 mg/kg that you
specified for lead is relatively high given the method used for its detection (ICP/MS), which would be
expected to have a significantly lower limit of quantitation. Because lead is not expected to be
present at quantifiable levels, a limit of 3 mg/kg may be unnecessarily high. In order to provide
assurance that lead will be present in your ingredient at minimal levels, we request that you reduce
the specified limit for lead to a level that more closely reflects the detection limit of the analytical
method, which would be expected to be much lower than 3 mg/kg.
We respectfully request a response within 10 business days. If you are unable to complete the
response within that time frame, please contact me to discuss further options.
If you have questions or need further clarification, please feel free to contact me or Shayla.
Thank you in advance for your attention to our comment.
Regards,
Lauren
From: Victoria <vdavis@prozure.net>

Sent: Sunday, September 26, 2021 6:53 PM
Cc: West-Barnette, Shayla <Shayla.WestBarnette@fda.hhs.gov>
Subject: [EXTERNAL] RE: GRN 957
CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you
recognize the sender and know the content is safe.
Thank you Lauren
Victoria Davis
Prozure
(561)926.2723
This email (and any attachments) is only intended for the use of the person or entity to which it is
addressed, and may contain information that is privileged and confidential. You, the recipient, are
obligated to maintain it in a safe, secure and confidential manner. Unauthorized disclosure or failure to
maintain confidentiality may subject you to federal and state penalties. If you are not the intended
recipient, please immediately notify us by return email, and delete this message from your computer.
From: Viebrock, Lauren [mailto:Lauren.Viebrock@fda.hhs.gov]

Sent: Friday, September 24, 2021 9:12 PM
To: Victoria
Subject: GRN 957
Hi Victoria,
Thank you and Michael for speaking with us regarding the lead specification limit provided in GRN
957. We will be in touch shortly with the written follow up question. The email may come from me,
but in the event that I am on leave at that time, it will come from my colleague Dr. Shayla West-
Barnette (whose contact information is copied on this email) and she will then become the
Regulatory Review Scientist and point of contact for your submission moving forward.
Best,
Lauren
Lauren VieBrock

Tel: 301-796-7454
Il l U.S. FOOD & DRUG

ADM IN ISUAT I ON
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GRAS Notice GRN 969 Bacillus Subtilis

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GRAS Notice GRN 969 Bacillus Subtilis

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GRAS Notice (GRN) No.

.Ai. AlBMR Life Sciences, Inc.

July 21, 2020

Susan Carlson, PhD SEP 1 6 '. 320

Dear Dr. Carlson:

Jessica Gruber, ND (agent of the notifier)

Bacillus subti/is Bss-19 GRAS 2

Dr. Carlson and staff,

Jared Douglas Brodin

Submitted by the Notifier:

Prepared by the Agent of the Notifier:

July 21, 2020

Part 1: Signed Statements and Certification ............................................................ 6

Bacillus sublilis Bss-19 GRAS 3

6.3 .1 Toxicological Studies on B. subtilis strains ......... .. ....................... .... ..... 29

Bacillus subti/is Bss-19 GRAS 4

Figures and Tables

Table 1. Genomic Average Nucleotide Identity Comparison of Bss-19 to Other Relevant

Bacillus subtifis Bss- 19 GRAS 5

Part 1 : Signed Statements and Certification

Agent of the Notifier

1.3 Name of the Substance

1.4 Intended Conditions of Use

Bacillus subtilis Bss-19 GRAS 6

additional regulatory review by USDA. The intended addition level to foods is up

1.5 Statutory Basis for GRAS Conclusion

1.6 Not Subject to Premarket approval

1.7 Data and Information Availability Statement

1.8 Exemption from Disclosure under the Freedom of

BacWus subtilis Bss-19 GRAS 7

Elizabeth McCartney Date

Bacillus subtilis Bss-19 GRAS 8

Part 2: Identity, Method of Manufacture, Specifications,

2.1.1 Taxonomy of Bss-19

2.1.2 Genetic Identification of Bss-19

Bacillus sublilis Bss-19 GRAS 9

Table 1. Genomic Average Nucleotide Identity Comparison of Bss-19 to Other

2.2.2 Raw Materials

2.2.3 Manufacturing Narrative and Flowchart

Bacillus subtilis Bss-1 9 GRAS JO

Bacillus subtilis Bss- 19 GRAS 11

After fermentation is complete, an additional aeration step is performed in the

Bacillus subtilis Bss-19 GRAS 12

Bacillus sublilis Bss-19 GRAS 13

Manufacturing Process Step

GLPs Approved Seed Culture QC Testing

HACCP,GMPs Seed Scale-up

HACCP.GMPs Commerical Fermentation

HACCP,GMPs Culture Concentration QC Testing

HACCP.GMPs Culture Spray Dried QC Testing

H ACCP,GMPs Metal Detection Standards Testing

HACCP.GMPs Culture Packaging

QC Release and Storage QC Testing

Figure 1. Manufactu ring Flowchart

Ta bl e2. Bss-19 s,pecifications

Bacillus subtilis Bss-19 GRAS 14

2.3.1 Batch Analysis

Ta bl e 3 Bss- 19 Bate h Ana1yses

Bacillus subtilis Bss- 19 GRAS 15

2.3.2 Residual Pesticide Analysis

2.3.3 Shelf-Life Stability

2.4 Antibiotic Resistance

Bacillus subti/is Bss-19 GRAS 16

Fallowing EFSA recommendations and guidelines, DuPont assessed the phenotypic

1. Kunst F, Ogasawara N, et al. The complete genome sequence of the gram