GRAS Notice GRN 990 Short Chain Fructooligosaccharides W Amendments

GRAS Notice (GRN) No.
990
https://www.fda.gov/food/generally-recognized-safe-gras/gras-notice-inventory
749 46th Square

Vero Beach, FL 32968, USA
Telephone: 772-299-0746
Soni &Associates Inc. Facsimile: 772-299-5381
E-mail: msoni@soniassociates.net
January 15, 2021
Paulette M. Gaynor, Ph.D.

Senior Policy Advisor h~~ ~ -,, ~[r, \ f[-::7
Office of Food Additive Safety (HFS-200)
Center for Food Safety and Applied Nutrition
Food and Drug Administration
JAN 2 5 2021 I
5100 Campus Drive FI . f
College Park, MD 20740 r .) AC _,If v E
Subject: GRAS Notification for use of short-chain Fructooligosaccharides in Infant

Formula
Dear Dr. Gaynor:
We respectfully submit the attached GRAS notice, on behalf of Tata Chemicals

Limited (India) for use of short-chain fructooligosaccharides (scFOS) in infant formula.
As regards submission of this GRAS notice, please note that on March 8, 2018, we had a
pre-GRAS meeting with Dr. Morissette and her team.
Based on the discussions with FDA and FDA recommendations (memorandum of
meeting- March 15, 2018), we have prepared the attached GRAS notice of a claim that
the use of scFOS in infant formula, described in the enclosed notification document is
exempt from the premarket approval requirement of the Federal Food, Drug, and
Cosmetic Act because it has been determined to be GRAS, based on scientific
procedures.
Please also note that the attached GRAS notice is a follow-up to Tata Chemical's
GRN 000605, which was for the intended use of FOS in conventional foods and received
a No Questions letter from FDA on March 17, 2016.
As required, please find enclosed three copies of the GRAS notification. If you
have any questions or require additional information, please feel free to contact me by
phone at 772-299-0746 or by email at sonim(albellsouth.net .
Sincerely,
Enclosure: Three copies of the GRAS notification
'N\'V',V .soniassociate;,.net
GENERALLY RECOGNIZED AS SAFE (GRAS) EVALUATION
OF SHORT CHAIN FRUCTO-OLIGOSACCHARIDES FOR USES
IN TERM INFANT FORMULA
Submitted by:
Tata Chemicals Limited
Innovation Centre
Ambedveth (V), Paud road, Mulshi,
Pune, Maharashtra - 412108
INDIA
Submitted to:
U.S. Food and Drug Administration
Office of Food Additive Safety
HFS-200
5100 Campus Drive
College Park, MD 20740
USA
December, 2020
GENERALLY RECOGNIZED AS SAFE (GRAS) EVALUATION OF
SHORT CHAIN FRUCTO-OLIGOSACCHARIDES FOR USES IN TERM
INFANT FORMULA
TABLE OF CONTENTS
1. Part I-SIGNED STATEMENTS AND CERTIFICATION ............................................... 4
1.1. Name and Address of Notifier ...................................................................................... 4

1.2. Name of Notified Substance .......................................................................................... 4
1.3. Intended Conditions of Use ........................................................................................... 4
1.4. Statutory Basis for GRAS Determination ................................................................... 4
1.5. Exclusion from Premarket Approval ........................................................................... 4
1.6. Availability of Data & Information .............................................................................. 5
1.7. Data Exemption from Disclosure ................................................................................. 5
1.8. Certification ...................................................................................................................... 5
1.9. Name, Position/Title of Responsible Person who Signs the Dossier and Signature. 5
1.10. FSIS/USDA - Use in Meat and/or Poultry .................................................................. 6
2. Part II - IDENTITY, SPECIFICATION, MANUFACTURING AND TECHNICAL

EFFECTS ....................................................................................................................................... 7
2.1. Identity ............................................................................................................................ 7

2.1.1. Description ...................................................................................................................... 7
2.1.2. Synonyms and Trade Names ........................................................................................ 7
2.1.3. Chemical Abstract Registry Number ........................................................................... 7
2.1.4. Chemical Formula and Molecular Weight .................................................................. 7
2.1.5. Chemical Structure ........................................................................................................ 7
2.1.6. Other Chemically Related Constituents ...................................................................... 8
2.2. Specifications .................................................................................................................. 8
2.3. Manufacturing Process ................................................................................................ 10
2.4. Technical Effects .......................................................................................................... 13
3. Part III - DIETARY EXPOSURE ........................................................................................ 14
3.1. Intended Use Levels and Food Categories ................................................................. 14

3.1.1. Estimated Daily Intake from the Proposed Uses ...................................................... 14
4. Part IV - SELF LIMITING LEVELS OF USE ................................................................... 16
Tata Chemicals Page 2 of63 FOS- IF-GRAS

5. Part V - EXPERIENCE BASED ON COMMON USE IN FOODS BEFORE 1958 ....... 17
6. Part VI - NARRATIVE ......................................................................................................... 18
6.1. DATA PERTAINING TO SAFETY .......................................................................... 18
6.1.1. Pivotal or Primary Published Clinical Studies of FOS in Infant ............................ 18

6.1.2. Secondary Published Studies ...................................................................................... 23
6.1.2.1. Studies in Infant with Similar Substances ................................................................. 23
6.1.2.2. Studies in Children and Adults ................................................................................... 26
6.1.2.3. scFOS Studies in Piglets and Other Weaning Animals ............................................ 26
6.1.2.4. Specific Animal Toxicity Studies of scFOS ................................................................ 30
6.1.2.4.1. Specific Acute Toxicity ............................................................................................... 30
6.1.2.4.2. Specific Dose-Range Finding Study .......................................................................... 30
6.1.2.4.3. Specific Subchronic Toxicity Study........................................................................... 31
6.1.2.5. Other Published Studies .............................................................................................. 32
6.1.2.5.1. Metabolism .................................................................................................................. 32
6.1.2.5.2. Toxicity Studies of FOS .............................................................................................. 33
6.1.2.5.3. Acute Toxicity Studies ................................................................................................ 33
6.1.2.5.4. Repeat-Dose Toxicity Studies ..................................................................................... 34
6.1.2.5.5. Developmental and Reproductive Toxicity Studies ................................................. 37
6.1.2.5.6. Mutagenicity and Genotoxicity Studies .................................................................... 38
6.1.3. Available Corroborative Safety Evidence ................................................................. 39
6.1.3.1. Regulatory Agency Review ......................................................................................... 39
6.1.3.2. Unpublished Corroborative Studies ........................................................................... 41
6.1.3.3. Natural Occurrence ..................................................................................................... 43
6.1.3.4. Current Uses ................................................................................................................. 44
6.1.3.5. scFOS Safety and Degree of Polymerization ............................................................. 44
6.1.3.5.1. Degree of Polymerization and Fermentability ......................................................... 44
6.1.3.5.2. Osmolality and Degree of Polymerization ................................................................ 46
6.1.3.6. scFOS and Changes in Colonic pH ............................................................................. 47
6.2. Summary, Discussion and Conclusion ............................................................................... 48
7. Part VII- SUPPORTING DATA AND INFORMATION ................................................. 53

1. Part I-SIGNED STATEMENTS AND CERTIFICATION
In accordance with 21 CFR § 170 Subpart E consisting of § 170.203 through § 170.285,

Tata Chemicals Limited (Tata), India hereby informs the FDA that short-chain fructo
oligosaccharides (scFOS), as manufactured by Tata, is not subject to the premarket approval
requirements of the Federal Food, Drug, and Cosmetic Act based on Tata's view that the notified
substance is Generally Recognized as Safe (GRAS) under the conditions of its intended use
described in Section 1.3 below.
1.1. Name and Address of Notifier
Bombay House, 24 Homi Modi Street,
Fort, Mumbai Maharashtra-400001
INDIA
1.2. Name of Notified Substance
The common name of the substance of this Generally Recognized As Safe (GRAS)
assessment is short-chain fructo-oligosaccharides (scFOS) or oligofructose. scFOS for food uses
will be marketed as standardized (to the content of FOS) powder. scFOS will be marketed under
the tradename- FOSSENCE™.
1.3. Intended Conditions of Use
Short-chain fructo-oligosaccharides (scFOS) is intended for use as an ingredient in non
exempt term infant formula at the maximum intended addition levels of 400 mg scFOS/100 ml in
starter formula (from birth to approximately 6 months) as consumed and 500 mg scFOS/100 ml
in follow-on formula (infants older than approximately 6 months) as consumed. FOS is not
intended for addition to pre-term formula. The intended uses and levels of scFOS in term infant
formula are identical to those described in GRN 537 (lngredion, 2014) and GRN 797 (NFBC,
2018). Based on energy intakes and the energy content of infant formula, the 90 th percentile
formula intake for males and females combined is estimated as 207 ml/kg body weight (bw)/day.
The 90 th percentile intake of scFOS is estimated as 828 mg/kg bw/day from starter formula
within the first month of life and about 800 mg/kg bw/day from the follow-on formula thereafter.
1.4. Statutory Basis for GRAS Determination
This GRAS conclusion is based on scientific procedures in accordance with 21 CFR
170.30(a) and 170.30(b).
1.5. Exclusion from Premarket Approval
Tata has determined that the use of scFOS derived from enzymatic conversion of sucrose
is Generally Recognized As Safe, under the conditions of its intended use in non-exempt infant
formula, consistent with Section 201(s) of the Federal Food, Drug, and Cosmetic Act. This
GRAS conclusion has been reached in accordance with requirements in 21 CFR 170.220.
Therefore, the use of FOS derived from enzymatic conversion of sucrose is exempt from the
premarket approval requirements of the FD&C Act.

1.6. Availability of Data & Information
The data and information that are the basis for this GRAS conclusion will be made
available to FDA upon request by contacting Mr. Dipak Bagad at the below addresses. The data
and information will be made available to FDA in a form in accordance with that requested
under 21 CFR 170.225I(7)(ii)(A) or 21 CFR 170.225(c)(7)(ii)(B).
Mr. Dipak Bagad
Manager - Regulatory Affairs
Innovation Centre, Tata Chemicals Limited
Ambedveth (V), Paud road, Mulshi,
Pune, Maharashtra - 412108
INDIA
Tel: +91- 020 66549772

Fax: +91-020 66549735
Email: dbagad@tatachemicals.com
1.7. Data Exemption from Disclosure
Parts II through VII of this GRAS notification do not contain data or information that is
exempt from disclosure under the Freedom of Information Act. There is no privileged or
confidential information such as trade secrets and/or commercial or financial information in this
document and the information contained in this dossier can be made publicly available.
1.8. Certification
Tata certifies that, to the best of its knowledge, this GRAS conclusion is based on a
complete, representative, and balanced dossier that includes all relevant information, available
and obtainable by TATA, including any favorable or unfavorable information, and pertinent to
the evaluation of the safety and GRAS status of the use of scFOS preparation. Tata accepts
responsibility for the GRAS determination that has been made for FOS derived from enzymatic
conversion of sucrose as described in this dossier.
1.9. Name, Position/Title of Responsible Person who Signs the Dossier and Signature
Mr. Rahul Gupta
Business Head - Nutritional Solutions
Mumbai, Maharashtra
INDIA
Tel: +91- 8976056249

Fax: +91- NA
Email: rgupta@tatachemicals.com
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1.10. FSIS/USDA - Use in Meat and/or Poultry
Tata does not intend to add scFOS to any meat and/or poultry products that come under
USDA jurisdiction. Therefore, 21 CFR 170.270 does not apply.

2. Part II - IDENTITY, SPECIFICATION, MANUFACTURING AND TECHNICAL
EFFECTS
Short-chain fructo-oligosaccharides (scFOS) are derived from food grade sucrose via a
transfructosylation catalyzed by P-fructofuranosidase enzyme derived from a non-pathogenic and
non-toxigenic strain of Aureobasidium pullulans.
2.1. Identity
2.1.1. Description
The scFOS product is white to light yellow syrup or off white to light yellow powder
with slight sweet taste and no odor.
2.1.2. Synonyms and Trade Names
FOS; Oligofructose; short-chain fructo-oligosaccharides (scFOS or FOS); Neosugar. The

systematic name of all fructans, including scFOS, is [et-D-glucopyranoside-(1-2)-]-P-D
fructofuranosyl-[ ( 1-2)-P-D-fructofuranosyl]n.
The subject of this GRAS assessment will be marketed under the trade name
FOSSENCE™.
2.1.3. Chemical Abstract Registry Number
The CAS Registry Number for fructo-oligosaccharides (FOS) is 308066-66-2.
2.1.4. Chemical Formula and Molecular Weight
The molecular formula for all fructans is C6H 11 O5 (C 6H 10 O5 )nOH. The formulas of its
three components are: 1-kestose - C 18 H32 O 16, nystose - C24 H42 O21 , and fructofuranosylnystose
C30H52O26. The molecular weight of individual three components of scFOS is as follows: 1-
kestose- 505 Da; nystose- 666 Da; and fructofuranosylnystose- 828 Da.
2.1.5. Chemical Structure
scFOS are a mixture of oligosaccharides consisting of a sucrose molecule (glucose -

fructose disaccharide, GFl) linked to one (GF2; degree of polymerization or DP3), or two
(GF3; DP4) or three (GF4; DP5) additional fructose units added by P2-l glycosidic linkages to
the fructose unit of the sucrose. Fructans can have degrees of polymerization (the number of
fructose or glucose residues) ranging from 2 to over 60. scFOS consists entirely of molecules
with degrees of polymerization between 3 and 5, consisting of 2 to 4 fructose residues and a
single terminal glucose residue. scFOS, the subject of this present GRAS dossier, primarily
consists of 3 different molecules, each containing a terminal glucose residue and 2, 3, or 4
fructose residues, designated as GF2, GF3, and GF4, also called as 1-kestose, nystose, and
fructofuranosylnystose, respectively. The structural formulas of 1-kestose, nystose, and
fructofuranosylnystose are shown in Figure 1.

b)
c) ~OH
a) H~ O H H I H
H H
H H OH H
HO HO
H OH H OH H O H
H<ko~
~
HO~ HO~O
H HO
H H
,H,
H
H
OH H CH2 OH H
OH H r2
Hoko~ uo~ Hk ~
r~~+'"
OH H OH
OH H l"' ~.
OH E-f IH2
!H)ko~ H<k ~
L ~ OH
~TH2 OH H
"'~
OH H OH
Figure 1. Chemical Structure of scFOS components (a) 1-Kestose (GFl), (b) Nystose (GF2), and (c)
Fructofuranosylnystose (GF3). Fructosyl units are linked at position P-2, 1 of sucrose.
2.1.6. Other Chemically Related Constituents

As described above, the subject of present GRAS assessment primarily contains small
chain fructo-oligosaccharides. Similar to scFOS, the longer chain chemically related fructans,
such as oligofructose and inulin, of P2-1 linked fructose molecules that may or may not have a
terminal glucose molecule are primarily derived by isolation and/or partial enzymatic hydrolysis
of inulin from chicory root. The term oligofructose has been typically used to characterize linear
oligosaccharides, ranging 3 to 6 saccharides in length. The term inulin is typically used to define
long-chain polymers of P2-1 linked fructose molecules with degrees of polymerization ranging
from 10 to 60 or more saccharides in length. These related polymers have similar chemical
composition to scFOS and are likely to have similar toxicological and physiological
characteristics following ingestion. These oligomers display a higher molecular weight
distribution. Given these differences between scFOS and other inulin type fructans, the subject of
this GRAS dossier has been primarily limited to discussion of scFOS produced from sucrose by
enzymatic synthesis. As some fructans product also contain relatively high levels of scFOS,
these products are also considered in this GRAS assessment.
2.2. Specifications
Food grade specifications of scFOS have been established by Tata Chemicals Limited
(Tata). scFOS for uses in infant formula will be marketed in the form of powder and liquid. The
specifications of scFOS-P95 and scFOS-L95 are presented in Table 1. To demonstrate
conformance with the food-grade specifications, Tata analyzed three batches of scFOS.
Analytical results from three lots (Appendix I) suggest that scFOS powder ((Appendix I A) as
well as liquid (Appendix I B) is consistently manufactured to meet the standard specifications.
The distribution ratio of scFOS components [1-kestose (GF2), Nystose (GF3) and
Fructofuranosylnystose (GF4)] for FOS-P95 and FOS-L95 is provided in Appendix I A and B.
The batch analysis data for scFOS demonstrate that the manufacturing process produces
oligomers that are characteristic of typical scFOS preparations derived from sucrose by
enzymatic action with GF2, and GF3 representing the major fructose oligomers and lower

quantities of longer chain GF4. The final product also contains small amounts (~5%) of residual
sucrose, glucose and fructose representing the major by products or residues in the ingredient.
The subject of this GRAS assessment, scFOS, is substantially equivalent to the scFOS that was
the subject of the GRAS notified substances for uses in infant formula, reviewed by the FDA
with no questions [including GRN 797 (NFBC, 2018) GRN 537 (lngredion, 2014) and GRN 44
(OTC Nutrition, 2000)].
Table 1. Food Grade Specifications of scFOS Powder (FOS-P9S) and Syrup (FOS-L9S)
Specifications
Parameters Method
FOS-P95 FOS-L9S
Fine white free flowing Colorless to sunshine
Description hygroscopic powder yellow color syrupy Sensory test
(clear in solution) liquid
Sweet, without foreign Sweet, without
Taste and Aroma Sensory test
tastes / odors foreign tastes / odors
FCC (Fructooligosaccharides
Total solids(%) NLT97.0 - short chain)
Moisture (Karl Fisher)(%) NMT 5.0 (w/w) NMT25 In-house
Brix {Refractometer) 0 Bx - NLT75 In-house
Residue on ignition FCC (Fructooligosaccharides
NMT0.l NMT0.1
(sulphated ash)(%) short chain)
pH (pH meter with 10%
5.0 - 7.5 5.0 - 7.5 In-house
solution @ 25°C)
Carbohydrate composition
(a) Identification
Fructose(% dry basis) NLT67.0 NLT67.0
Glucose (% dry basis) NMT 33.0 NMT33.0
(b) Assay
Total
NLT 95.0 NLT95.0 FCC (Fructooligosaccharides
Fructooligosaccharides (%)
short chain)
-- Trimer (GF2) Informative Informative
-- Tetramer (GF3) Informative Informative
-- Pentamer and larger (GF4
Informative Informative
and higher)
(c) Sucrose + Glucose
NMT5.0 NMT5.0 AACC 80-04.01
+ Fructose
Heavy metals
SO-IN-MUL-TE-063A By
Lead (as Pb) (ppm) NMT0.02 NMT0.02
ICPMS
Arsenic (as As2O3) (ppm) NMT0.1 NMT0.l
ICPMS
Cadmium (Cd) (ppm) NMT0.01 NMT0.Ql
ICPMS
Mercury (as Hg) (ppm) NMT0.01 NMT0.Ql
ICPMS
Chromium (as Cr) (ppm) NMT0.05 NMT0.05 SO-IN-AFL-MNR-C-TE-006
Tin (as Sn) (ppm) NMT50.0 NMTS0 SO-IN-AFL-MNR-C-TE-006
Copper (as Cu) (ppm) NMT30.0 NMT30 SO-IN-AFL-MNR-C-TE-006

Table 1. Food Grade Specifications of scFOS Powder (FOS-P95) and Syrup (FOS-L95)
Specifications
Parameters Method
FOS-P95 FOS-L95
Methyl Mercury (Calculated
NMT0.25 NMT0.25 SO-IN-AFL-MNR-C-TE-006
as the element) (ppm)
Microbiological limits
Total Plate Count (cfu/g) NMT300 NMT 300 IS 5402 : 2012
Enterobacteriacea (MPN/g) NMT3 NMT3 IS 7402
Yeasts & Mould (cfu/g) NMT20 NMT20 IS 5403 :1999 (Reaff.2013)
IS 5887 (Part I) I 976 (Reaff.
Escherichia coli (MPN/g) Absent 10 g Absent 10 g
2013)
IS 5887 (Part II) 1976 (Reaff.
Staphylococcus aureus Absent 10 g Absent 10 g
2013)
IS 5887 (Part III) 1999 (Rea:ff.
Salmonella spp Absent 100 g Absent 100 g
2013)
IS 5887 (Part VII) 1999
Shigella spp Absent25 g Absent25 g
(Rea:ff. 2013)
Listeria monocytogenes Absent25 g Absent25 g ISO 11290 (Part I) 2017
Sulphite reducing Clostridia
NMTlO NMT 10 ISO 15213: 2003
(cfu/g)
Cronobacter sakazakii Absent300 g Absent300 g ISO 22964: 2017
IS 5887 (Part VI) 1999 (Rea:ff.
Bacillus cereus (cfu/g) NMT 100 NMT 100
2005)
Mycotoxins
Aflatoxin Bl (ppb) NMT0.5 NMT0.5
Aflatoxin B2 (ppb) NMT0.5 NMT0.5 AOAC 999.07 by HPLC using
immunoaffinity column and
Aflatoxin GJ(ppb) NMT0.5 NMT0.5 Kobra cell
Aflatoxin G2 (ppb) NMT0.5 NMT0.5
Aflatoxin Ml (ppb) NMT0.025 NMT0.025 SO-IN-AFL-MNR-C-TE-065
SO-IN-AFL-MNR-C-TE-023
Melamine {ppm) NMT0.5 NMT0.5
(Ref: USFDA)
NL T = Not less than; NMT = Not more than; CFU-;; Colony forming units
2.3. Manufacturing Process

scFOS (FOSSENCE™) is produced by the action of microbial enzyme; ~
fructofuranosidase/fructosyltransferase on sucrose syrup. P-Fructofuranosidase is an intracellular
enzyme produced by a wild type (natural) strain of the fungus, Aureobasidium pullulans,
referred here as Culture. The microorganism (A. pullulans) used in the production of scFOS,
which is intended to be used in infant formula is the same that has been used in the
manufacturing of scFOS, subject of GRN 605 that received no question letter from FDA for the
use in conventional foods. In the food industry, A. pullulan is used in the production of food
ingredients. A. pul/ulan is used in the production of pullulan. A. pul/ulans used in the production
of scFOS is non-toxigenic and non-pathogenic and is registered under the Microbial Type
Culture Collection (MTCC), Chandigarh in India with accessions no MTCC 5490. The
production process of scFOS is developed by using whole cell microbial biotransformation
technique utilizing the membrane bound enzyme; f3-Fructofuranosidase. The production process
of the FOS contains three major steps:

1. Microbial fermentation for the production of cell biomass, which is used as source of
enzyme this process also referred as Upstream Process (USP).
2. Sugar Solution Preparation & Biotransformation of sucrose to FOS (BT)
3. Purification and Concentration ofFOS or Downstream Process (DSP).
1. Microbial fermentation for the production of enzyme and cell biomass or Upstream
Process (USP):
In the first step of FOS production, microbial Culture (biomass) is generated by
fermentation technique by inoculating of seed-culture in the main fermenter. The Lyophilized
vial (master culture) from culture bank is used to prepare a mother culture from which the stock
culture is prepared subsequently. These stock cultures are stored at -80°C and used to prepare the
working cultures for the production. The culture is prepared using growth media consisting of
anhydrous glucose, yeast extract powder, peptone and polypropylene glycol (antifoaming agent).
Flasks with the media are inoculated and incubated at 27-28°C for the period 24-48 hours. In
addition, the culture is also inoculated into sterile nutrient broth (NB) tubes for sterility checking,
that are incubated at 37°C. Following confirmation of desired growth pattern and purity (i.e., no
contamination), the culture is used as the seed inoculum for the next stage. The quantity of the
microbial biomass is built up to the production level through successive stages of culturing of the
seed culture, verifying at each step for desired characteristics and purity. On the last step, the
growth medium is slightly modified to include sucrose in order to stimulate the production of the
intracellular enzyme by the microbial culture. The biomass required for production is separated
using a plate and frame filter press under sterile conditions. The separated biomass is kept frozen
(-80 °c) till needed for production.
2. Sugar Solution Preparation & Biotransformation (BT):
In the second step, initially prepared the sugar solution from the purified cane sugar (50
brix) and pasteurized at 72°C for 30 seconds. The generated biomass of Culture-A is then
reacted with the sugar solution (sucrose 50%) in a bioreactor and the reaction is carried out at
optimum temperature, pH & agitation speed. The progress of the reaction is monitored with the
help of HPLC by analyzing the reaction mixture at various time intervals. The reaction is
terminated after complete conversion of sucrose to FOS which would be around 55 to 60%. The
termination of reaction is carried by heating whole fermentation broth at 80°C for 1 hours, this
treatment inactive the enzyme and culture biomass. Subsequently, biomass is harvested by
filtration with filter press and the clear filtrate is subjected to downstream operations.
3. Purification and Concentration of FOS or Downstream Process (DSP) of the FOS.
Recovered dilute, partly pure FOS solution from the biotransformation step is subjected
to various downstream processing operations for the purification and concentration purpose as
mentioned below:
A. Activated Carbon Treatment
B. Resin Treatment
C. Chromatography treatment

D. Polishing treatment
E. Concentration and Pasteurization
F. Spray drying and packaging
A. Activated carbon treatment:
Activated carbon treatment is carried out to remove color and organic impurities generated
during the biotransformation process. In this process activated carbon is added in the enzyme
inactivated FOS solution and the mixture is stirred for the 4 hours at 60°C and then carbon is
separated from the FOS solution with help of plate filtration system. Subsequently FOS
solution is filtered through 0.2 micron filtration to removes carbon traces.
B. Resin treatment:
Resin treatment is carried out by using ion exchange resins for the removal of color, organic,
metal and mineral (ash) impurities. There are two types of the food grade polymeric resins;
cations and anions are used for the treatment. Both the resins are filled in a mixed bed resin
column and the column is regenerated with acid and alkali solutions. The FOS solution
collected after carbon treatment is passed from the column at 40°C temperature and
subsequently collected in a clean tank.
C. Chromatography treatment:
The resin purifies FOS solution is further passes through the chromatography column, which
is filled with the gel filtration type food grade polymeric resins to improve the FOS content
and remove other saccharides. The outcome of the chromatography will be more than 95%
purity FOS solution.
D. Polishing treatment:
The polishing treatment is carried out by using mixed bed resin column, which is filled with
anionic and cationic food grade polymer resins, which further removes the traces of organic,
metal and mineral (ash) impurities. The resin treatment is carried out at 40 °c temperature.
The clean FOS solution is further passed through 0.2 micron polish filter to remove any of
the fine particles and contaminants.
E. Concentration and pasteurization:
Concentration is mainly carried out to remove the water content of the liquid dilute FOS
solution collected after polishing step. FOS solution is subjected to vacuum evaporation
system and concentration is performed at 65°C to achieve the brix of the solution to 50% &
75%, for the manufacturing of powder product and liquid products, respectively. The
concentrated solution is then pasteurized at 76°C temperature for 15 seconds with the help of
heat exchanger, which ultimately reduces the microbial contaminations to the accepted levels.
F. Spray drying and packaging:
The concentrated and pasteurized 50 brix solutions is finally spray dried in a spray drier and
maintained the moisture content below 3% to reduce the possibility of lump formation and
microbial contaminations. The powder product is then packed in a clean 20 Kg Nylon bag.
Whereas the liquid product is packed in 25 Kg Jerry Cans, made up of food grade containers
(HDPE). The final product is then stored in dry place at ambient temperature.

Process Flow Diagram - Upstream Process -
~
Master Culture Stock Culture Seed Culture Biomass
(Lyophilized) ~ (-80°C) (27-28°C) Production
..JJ..-
Termination of Filtration &
Reaction (80°C) <=3 Biotransformation
(45°C)
Sugar Preparation
& Pasteurization <=3 Storage (-20°C)
~
Biomass Filtration
c:>I FOS Solution
Process Flow Diagram - Downstream Process -
Activated Carbon Filtration & Polishing Filter Resin Treatment

Treatment Clarification
Liquid Product Evaporation & Polishing Resin & >--. Chromatography

(75 brix) Pasteurization Fine Filtration ~ Purification
Packaging (HDPE) Spray Drying &

Containers Packaging
All raw materials and processing aids used in the manufacturing process for scFOS such
as hydrochloric acid, sodium hydroxide, and activated carbon, are suitable, food grade, and are
used in accordance with current good manufacturing practices. Hydrochloric acid and sodium
hydroxide are GRAS for use in food production, limited only by current Good Manufacturing
Practice (21 CFR §182.1057 and §184.1631, respectively). Food-grade activated carbon is an
unlisted GRAS substance with a long history of safe use in food processing. The resins and
microfiltration used are in compliance with FDA guidelines. The manufacturing facility 1s
certified with FSSC 22000 (Version 5) (2020/23).
2.4. Technical Effects
Tata intends to add scFOS to infant formula in order to enhance the organoleptic
properties and palatability of formula, and to provide a non-digestible oligosaccharide that may
improve stool consistency, reduce the risk of constipation, serve as a source of colonic
fermentation, and modulate colonic bacterial colonization in the infant receiving the formula
containing scFOS.

3. Part III - DIETARY EXPOSURE
3.1. Intended Use Levels and Food Categories

Tata intends to use scFOS in non-exempt infant formula at maximum addition levels of
400 mg/100 ml in starter formula (from birth to approximately 6 months) as consumed and 500
mg/100 ml in follow-on formula (infants older than approximately 6 months) as consumed.
3.1.1. Estimated Daily Intake from the Proposed Uses
The proposed uses of scFOS, by Tata, as a food ingredient in term infant formula and
follow-on formula at use levels 400 and 500 mg/100 ml of formula as consumed, respectively,
are identical to those described in the previous GRAS notices that received no question letter
from FDA. The resulting exposures of scFOS from its proposed uses have been estimated in the
previous GRAS notices (GRN 797; GRN 537) submitted to FDA. The scFOS product described
by Ingredion (2014) in GRAS notice (GRN 537) and NFBC in GRAS notice (GRN 797) was
reported to contain 95% of scFOS. The subject of this present GRAS notice also contains the
same levels of FOS. Furthermore, composition of the three primary constituents of scFOS ( 1-
Kestose, Nystose and Fructofuranosylnystose) in the subject of present GRAS notification is
substantially equivalent to the subject of GRN 537 (Ingredion, 2014) and GRN 797 (NFBC,
2018).
In determining the FOS intake, Ingredion (2014) in GRN 537 considered daily energy
intake of formula fed children. In these estimates of intake, daily energy consumption of infants
fed infant formula provided by Femon (1993) were considered. The subpopulation of infants,
boys in the age range 14-27 days, were found to have the highest intake of energy per kg body
weight. The 90 th percentile energy intake in this age group was reported as 141.3 kcal/kg bw/day.
The highest energy intake in girls in the same age group, 14-27 days, was reported as 138.9
kcal/kg bw/day that was similar to boys. In order to represent extreme intake, the FDA typically
uses the 90 th percentile of the intake distribution. In a 2008 Feeding Infant and Toddler Study by
Butte et al. (2010) further corroborated the energy intake estimates reported by Femon (1993). In
the study by Butte et al. (2010), the reported 90th percentile energy intake of 779 kcal or
approximately 144 kcal/kg bw is similar to the estimates reported by Fomon (1993).
The available information suggest that majority of the standard ready to consume
formulas contain 67 kcal/100 ml. In order to obtain 141.3 kcal energy/kg bw, an infant boy must
consume 209 ml formula/kg bw. Similarly, for an infant girl, to reach her 90 th percentile of
energy consumption of 138.9 kcal/kg bw/day, she will need to consume 205.5 ml formula/kg bw.
Based on these values, the 90th percentile of formula intake for the two sexes combined will be
about 207 ml formula/kg bw/day. Based on these assumptions, the 90th percentile daily intake of
FOS, added at a maximum concentration of 400 mg/100 ml to the starter formula is estimated to be
828 mg/kg bw/day. Similarly, the 90 th percentile daily intake of FOS from follow on formula
(containing 500 mg FOS/100 ml) is estimated as 1035 mg/kg bw/day. It should be noted that by the
time follow on formula is introduced, consumption of infant formula (on a body weight basis)
has decreased by about 20% and, even though the maximum intended addition level of scFOS is
increased to 500 mg/100 ml, the 90 th percentile intake of scFOS is only about 800 mg/kg bw/day.
It is recognized that as the infant grows, formula intake increases, but more slowly than
weight gain, so that consumption assessed as ml formula per kg body weight is lower for infants

older than 27 days. As a result of this and as the infant grows, intake of scFOS per kg body
weight decreases. The estimated intake of scFOS at 90th percentile peaks at about 1035 mg/kg
bw/day during the first 6 weeks of life, then begins to decline and by weeks 8-12, it reaches to
approximately 840 mg/kg bw/day. This suggest that the maximum estimated daily intake (EDI)
of FOS is unlikely to exceed 1035 mg/kg bw/day.
For long-term exposure, the assumptions made in these estimates are quite conservative,
because as the infant grows the formula intake increases but at a slower rate than weight gain.
Historically non-exempt infant formulas provided 20 kcal/fl Oz as fed. However, recent
information indicates that several infant formula notifications provide only 19 kcal/fl Oz. Given
this, at a maximum use level of 500 mg scFOS/100 ml of infant formula, and maintaining the
same energy intake, the 90 th percentile daily intake of scFOS would increase approximately 54.5
mg scFOS (increase from 1035 mg/kg bw/day to 1090 mg/kg bw/day) with consumption of the
lower 19 kcaVlO0 ml infant formula. As an infant grows and starts consuming complimentary
foods , and thus reduces the intake of infant formula, the level of scFOS consumed will decrease
due to the complimentary foods that are unlikely to contain the same level of scFOS as infant
formula.
In summary, the proposed use level of scFOS in starter formula is 400 mg/I 00 ml
formula, resulting in a 90 th percentile intake of 828 mg scFOS/kg bw/day during the period from
14 to 27 days of age, the period of highest formula intake. By the time follow-on formula is
introduced, consumption of infant formula (on a body weight basis) has decreased by about 20%
and, even though the maximum intended addition level of scFOS is increased to 500 mg/I 00 ml,
the 90 th percentile intake of scFOS is only about 800 mg/kg bw/day. For safety assessment
purposes of scFOS in infant formula, the maximum intake of 828 mg scFOS/kg bw/day is
considered.

4. Part IV - SELF LIMITING LEVELS OF USE
There are no known self-limiting levels of use that are associated with the use of notified
ingredient scFOS.

5. Part V - EXPERIENCE BASED ON COMMON USE IN FOODS BEFORE 1958
Not applicable. The statutory basis for the conclusion of GRAS status of scFOS in this
document is based on scientific procedures and not based on common use in food before 195 8.

6. Part VI - NARRATIVE
Non-digestible oligosaccharides, including FOS, have received considerable attention in
recent year for their potential health effects such as reducing constipation, decreasing levels of
serum lipids (cholesterol, triacylglycerols, phospholipids), stimulating growth of bifidobacterial
in the human colon, improving mineral absorption, as a non-cariogenic, and as a low-calorie
sweetener, etc. As a result of these properties of oligosaccharides, FOS is increasingly included
in food products and infant formulas. Given their potential health benefits and increased uses in
foods, scFOS have been extensively investigated for its safety and efficacy. The toxicity
potential of FOS have been summarized in several published experimental studies and review
articles. These studies include short and long-term toxicity studies in experimental animals,
metabolic (in vitro and in vivo) experiments, and human clinical studies, including studies in
infants. Additionally, the safety in use of FOS have been extensively and critically evaluated by
national and international regulatory agencies such as the FDA, FSANZ, and EFSA (SCF).
These agency reviews demonstrate that FOS is safe for its intended use as an ingredient in food,
including infant formula.
In the published literature, several preclinical and clinical studies with FOS have
appeared. In the following section, relevant efficacy and toxicological studies on FOS are
summarized in support of the conclusions drawn in this GRAS assessment. Efforts have been
made to present both the data supporting the safety as well as any data on the adverse effects of
FOS. In this GRAS assessment, attempts have been made to summarize the available
information, related to safety of FOS, in the order of their importance. First, the published
pivotal studies are described, followed by secondary published studies, then corroborative
unpublished studies and finally regulatory agencies assessments are summarized. The safety in
use of the proposed use of scFOS in infant formula is based on the totality of available evidence.
6.1. DATA PERTAINING TO SAFETY
6.1.1. Pivotal or Primary Published Clinical Studies of scFOS in Infant
The available pivotal studies of scFOS in infants related to growth and safety are
summarized in Table 2 as well as further described below.
In a prospective, randomized, double-blind study, Guesry et al. (2000; published as
abstract; also described in GRN 53 7) investigated the effects of 3 doses of scFOS in infants. In
this study, 53 infants (age- 7 to 20 day old) were randomized to receive five bottles of formula
per day for two weeks. Each bottle provided either 200 mg lactose or 200, 400, or 600 mg scFOS
providing daily intakes of 1 g lactose or 1, 2, or 3 g scFOS. As, the volume of the formula in
each bottle was not stated, the dietary concentration of FOS in mg/ml could not be determined.
However, the actual intake of FOS was reported. The infants were examined and weighed
weekly and mothers were asked to record daily formula consumption, stooling patterns, diaper
rash, spitting up, vomiting, or other events. Stool samples were collected at baseline, at the end
of feeding period, and two weeks later for pH measurement and enumeration of bifidobacteria.
Drop-out rates did not differ by group. A dose-related increase in stooling frequency with scFOS
intake was observed. There were no differences in bifidobacterial counts, fecal pH, or adverse
effects. Assuming that the infants were of normal weight for this age range, they would have
averaged about 3.7 kg; this level of intake would provide 811 mg scFOS/kg bw/day. This
amount is the mean daily intake of FOS that would result from addition of 680 mg scFOS/100 ml

formula. The findings from this study support the safety in use of scFOS in infant formula
proposed by Tata.
Table 2. Pivotal or Primary Studies of scFOS in Infants
Dose, Duration Study Design,

Subjects Results Reference
Obiective
scFOS - 200, 400, Prospective, 53 infants Drop-out rates did not differ by group. Guesry
or 600 mg/day randomized aged 7- Stooling frequency increased dose- et al.
for 2 weeks double-blind 20 days dependently with scFOS intake. There (2000)
study comparing were no differences in fecal pH,
the effects of 3 bifidobacteria counts, or adverse effects
concentration
levels of scFOS
in infant formula
scFOS-0 or Randomized, 186 healthy There were no significant differences between Lasekan et
2.5 g/L formula double-blind, term infants formula groups in completion rates, formula al. (2015)
until 35 days of placebo- aged 0- 8 intake, growth. stool frequency or consistency,
age controlled, days feeding-associated spit-up or vomit, urine
multicenter specific gravity, hydration status, adverse
study of events, or serious adverse events. Two serious
tolerance to soy- adverse events were reported in each formula
based infant group, but all were considered not study
formulas with related. The authors concluded that, ''This
scFOS and study demonstrated that the addition ofFOS at
mixed 2.5 g/L and mixed carotenoids to soy protein-
carotenoids based formulas, with or without sucrose, was
safe and well tolerated in healthy term
newborn infants."
scFOS - 0, 2.4, or Randomized, 97 healthy Dropouts from each group were: Control Xia et al.
3.4 g/L formula double-blind, term group-IO drop-outs, 1 due to parental (2012)
for 4 weeks placebo- infants report of intolerance; 2.4-g scFOS
controlled, aged~6 group-11 drop-outs, 3 due to parental
multicenter days(mean report of intolerance, 2 withdrawn by
study of the =2.3±0.3 investigators due to non-test-article related
effects of days) adverse events; 3.4 g scFOS group - 6
feeding on the drop-outs, 1 due to parental report of
intestinal intolerance.
microbiota
No differences were reported among groups
in stool frequency or consistency, frequency
of feedings with spit-ups or vomit, or total
bacterial loads. The highest abundance of
bifidobacteria was in the high-scFOS group,
but differences among groups were not
significant. Lactobacilli, bacteroides, E. coli,
and C. difficile levels were not significantly
different across groups. The authors
concluded that infant formula is similar to
human milk in its ability to support
bifidobacteria and lactobacilli, but suggested
that ..future improvement of infant formula
should be directed to reduce the abundance of
potentially harmful bacteria including E. coli

and C. difficile."
scFOS 5 g/L for 6 Prospective, 75 healthy 81 % of the infants suffered adverse events, Ripoll et
months randomized, 4-month- but there were no significant differences al. (2015)
double-blind, old infants between groups receiving scFOS or
placebo- maltodextrin placebo; few were regarded as
controlled, feeding-related and these did not differ
multi center between groups. No differences were
study of the observed between groups in the incidence or
effect of scFOS severity of intolerance symptoms, growth
on growth, (weight and height), or secretory IgA levels.
digestive A significant! y greater number of fecal
tolerance, fecal bifidobacteria was noted in the scFOS group
bifidobacteria as compared to controls after one month of
count, and feeding, but the difference was no longer
specific significant after 2 months. The authors
poliovirus concluded that, "The overall digestive
secretory IgA tolerance of the scFOS supplemented follow-
on milk formula is very good and confirms
that scFOS can be used safely at 5 g/L in
infants older than 4 months."
scFOS 4 g/L to age Prospective, 61 healthy Formula consumption and growth did not Paineau et
4 months randomized, term infants differ between the group receiving scFOS and al. (2014)
double-blind, aged 0-7 a control group that received maltodextrin.
placebo days (mean There was no difference in incidence or
controlled, age= severity of adverse effects between groups.
multicenter trial 4.1±0.8 Fecal bifidobacteria counts were significantly
of effect of days) higher among infants receiving scFOS than
scFOS on those receiving maltodextrins, but no
bifidogenesis significant difference was seen in poliovirus-
and specific IgA. The authors concluded that,
antipoliovirus "This study demonstrates that a milk- based
IgA infant formula supplemented with scFOS at 4
g/L will increase the fecal content of
Bifidobacteria in healthy term infants in
comparison to a placebo formula without
inducing any problem of digestive tolerance."
In a parallel feeding randomized, double-blind, 28-day trial in healthy term newborn

infants, Lasekan et al. (2015) compared the effects of soy-based infant formulas containing
supplemental scFOS on gastrointestinal (GI) tolerance and hydration. In this study, the infants
were fed either a commercialized soy formula (with history of safe use) containing sucrose as
20% of total carbohydrate, no supplemental scFOS and no mixed carotenoids (lutein, lycopene,

beta-carotene) as a control (CF, n=62 infants) or 1 of 2 experimental soy-based formulas, EFl
(n=64) and EF2 (n=62) containing scFOS (2.5 g/L) and mixed carotenoids (lutein = 53 µg/L,
lycopene = 81 µg/L and beta-carotene= 30 µg/L). EFl differed from EF2 by containing sucrose.
Although the degree of polymerization of supplemental FOS was not described, the investigators
clearly stated the use of scFOS. No significant study group differences in study completion rates
(CF=81, EF1=86, and EF2=87%), growth, stool frequency, formula intake, spit-up/vomit, mean
rank stool consistency, and safety measures (urine specific gravity, USG; hydration status and
adverse events) were noted.
In the study by Lasekan et al. (2015), a total of six serious adverse events were reported,
two in each study group and were rated by investigators as "not related" or "probably not
related" to the study formulas. The number of parental reports of loose/watery stools in the CF,
EFl and EF2 were 4, 7 and 2, respectively. However, these were not significantly different and
the hydration status and urine specific gravity for these subjects were normal. The findings from
this study suggest that term infants fed soy-based formulas supplemented with scFOS and mixed
carotenoids, with or without sucrose in the first 35 days of infancy showed good tolerance and
hydration that was comparable to the control soy-based formula with history of safe use. The
investigators also noted that a higher level of scFOS may be needed to produce a softer stool
consistency. The findings from this study did not reveal any adverse effects of scFOS. The use
levels of scFOS used in this study is lower (250 mg/ 100 ml) as compared to proposed uses by
Tata.
Xia et al. (2012) analyzed intestinal bacterial populations from term infants fed formula
supplemented with FOS. In this randomized, double-blind, placebo-controlled 4-week trial,
healthy term infants aged :S 6 days were enrolled to investigate the effects of four types of
feeding on the intestinal microbiota. The types of feeding included cow's milk (control), two
FOS groups (240 or 340 mg FOS/100 ml), and human milk (reference). Although the publication
mentioned use of FOS and not scFOS, the available information from other sources suggest that
the test article used was scFOS. A total of 65 infants completed the study. No differences were
reported among groups in stool consistency or frequency, or in the frequency of feedings with
spit-ups or vomit. The groups did not differ in total bacterial loads, although they tended to be
lower in the infants fed human milk as compared to formula-fed infants. The investigators
concluded that infant formula is similar to human milk in its ability to support bifidobacteria and
lactobacilli, but suggested that future improvement of infant formula should be directed to reduce
the abundance of potentially harmful bacteria including E. coli and C. difficile. The results of this
study support the safety of scFOS at the maximum use levels of up to 340 mg/100 ml. Although
the use levels of scFOS in this study are lower as compared to the present GRAS, the findings
did not reveal any adverse effects related to scFOS.
In a randomized, controlled, double blind trial, Ripoll et al. (2015) studied the effect of
scFOS on digestive tolerance and growth parameters in infants up to 10 months of age. In this
study, 75 formula-fed healthy infants were enrolled at the age of four months received either a
placebo or scFOS supplemented formula for six months. Infants meeting all eligibility criteria
were randomized (1:1 ratio) either in the scFOS group (follow-on milk formula supplemented
with scFOS at 500 mg/100 ml - 3.5% in replacement of maltodextrins in the powder) or in the
control group (follow-on milk formula without scFOS supplementation). Fecal poliovirus slgA
after vaccination and bifidobacteria concentration, weight, height, and digestive tolerance (i.e.,

constipation, crying, soft stool, vomiting and regurgitation, adverse events and serious adverse
events) were monitored.
In the study by Ripoll et al. (2015), tolerance and growth parameters were similar in both
the groups. Overall, 81 % of infants experienced at least one adverse event, with no significant
difference in the number of adverse events between groups. The most prevalent adverse event in
all infants were bronchitis (12%), gastroenteritis (9%), and nasopharyngitis (28%). No difference
was observed between groups for diarrhea and gastroenteritis. During the study, six different
infants suffered from serious adverse events. None of the serious adverse event was related to the
study product. Digestive tolerance was evaluated during the six month-study for infants who
received at least one feeding of follow-on milk per day, (equivalent to at least 2.5 g/day). There
was no difference between the two groups in terms of prevalence of digestive symptoms except
for the number of days with vomiting that was lower and the number of days with soft stools that
was higher in the scFOS group. The investigators reported that after six months of
supplementation, the strict follow-up of adverse events and digestive tolerance criteria have
demonstrated the good tolerance of scFOS follow-on milk, as no difference was observed
between groups for gastroenteritis, constipation, diarrhea, prevalence of infections, regurgitation,
and crying while these conditions are common at this life-stage. The authors also noted that
infants consuming the scFOS supplemented formula have experienced an improvement in
vomiting prevalence and in stool consistency.
The results of Ripoll et al. (2015) study show that a follow-on milk formula
supplemented with 500 mg/100 ml scFOS is safe and well tolerated leading to normal growth in
infants after the age of four months and promotes fecal bifidobacteria levels after one month in
infants who had never been breast-fed. scFOS addition elicited normal digestive tolerance and
normal growth suggesting it can be used safely at 500 mg/100 ml in infants after four months of
age. The findings from this study support the proposed use of scFOS in follow on formula by
Tata. Ripoll et al. (2015) also suggested that findings from their study (described above)
compliments the data from previous studies by Euler et al. (2005) and Veereman-Wauters et al.
(2011) that revealed no negative impact on growth following supplementation with FOS
(oligofructose from chicory- by partial enzymatic hydrolysis) at dose from 3 to 8 g/L in younger
infants after 4 and 5 weeks of supplementation.
In yet another randomized, double-blind, placebo-controlled trial, Paineau et al. (2014)
investigated the effects of scFOS on fecal bifidobacteria and specific immune response in
formula-fed infants. In this study, 61 healthy term infants aged 0-7 days (mean age=4.1±0.8
days) were allocated to receive formula supplemented with 400 mg/100 ml of either scFOS or
maltodextrins until the age of four months. The scFOS used had a degree of polymerization
between 3 and 5 that is substantially equivalent to the scFOS that is the subject of this GRAS
notice. Stool samples were collected prior to clinic visits at baseline and at the ages of 2, 3, and 4
months for analysis of bifidobacteria and antipoliovirus IgA. Additionally, weight and length of
the infant were also measured at each clinic visit. Parents were asked to maintain diaries on
formula consumption, digestive tolerance (assessed by incidence of abdominal pain, diarrhea,
and vomiting), and adverse effects.
In the study by Paineau et al. (2014), the amount of formula consumed did not differ
between the groups, nor did growth. The most frequent adverse event was abdominal pain,
followed by liquid stools without any difference in incidence or severity between the feeding
groups. Only one serious adverse event of an episode of bronchitis unrelated to feeding was

reported. In infants receiving scFOS, fecal bifidobacteria counts were significantly higher as
compared to receiving maltodextrins, but no significant difference was seen in poliovirus
specific IgA. The investigators concluded that the findings from this study demonstrates that a
milk-based infant formula supplemented with scFOS at 400 mg/100 ml will increase the fecal
content of bifidobacteria in healthy term infants in comparison to a placebo formula without
inducing any problem of digestive tolerance. The findings from this study support the safety and
tolerance of formula containing scFOS at levels of 400 mg/100 ml in infants and are applicable
to the present GRAS.
In a prospective, interventional open label trial, Vandenplas et al. (2017) investigated the
effects of a new symbiotic infant formula, supplemented with Bifidobacterium lactis and FOS,
with lactose and a whey/casein 60/40 protein ratio, administered to 280 infants for three months.
The study formula was added with FOS (350 mg/100 ml) and B. lactis (10 7 cfu/g powder). The
inclusion infant in the study was based on parents who intended to feed their infants (partially)
formula and agreed to feed them the new symbiotic formula. The degree of polymerization for
FOS was not mentioned in the study. The age of infant at entry was 3.8 ± 3.6 weeks. Of the 280
infants, 75 received the study formula from birth and 227 infants fed during the trial period
received the study formula exclusively. The median age of the infants at inclusion was 0.89
months. Weight 'evolution' (as mentioned in publication) was in accordance with the World
Health Organization growth charts for exclusive breastfed infants.
In the study by Vandenplas et al. (2017), the measurement of all anthropometric
parameters (weight-for-length z score and body mass index-for-age z score) was within the
normal range. The incidence of daily regurgitation (10.9%), infantile crying and colic (10.5%),
and functional constipation (3.2%) were all significantly lower as compared to the reported
median prevalence for a similar age according to the literature (median value of 7.8% for
functional constipation, 26.7% for regurgitation, 17.7% for infantile colic). No serious adverse
event related to the study product was reported. The investigators concluded that new symbiotic
infant starter formula (containing 0.35 g FOS/100 ml) was safe, resulted in normal growth and
was well tolerated. The results of this study support the safety of scFOS at use levels ofup to 350
mg/100 ml.
In summary, the available studies in infants suggest that levels up to 680 mg scFOS/100
ml of infant formula is well tolerated by infants without any adverse effects. The test articles
used in the above described studies is substantially equivalent to the subject of present GRAS.
The minor differences in the scFOS product is unlikely to cause any difference in toxicological
or clinical effects. Thus, the clinical evidence from the above described studies is applicable to
the current scFOS. The findings from these studies support the proposed uses of scFOS by Tata
in term infants as stated in this GRAS assessment.
6.1.2. Secondary Published Studies
6.1.2.1. Studies in Infant with Similar Substances
In the published literature there are several studies with oligofructose (FOS) derived from
other sources such as chicory. These studies are considered as secondary pivotal studies as the
molecules are similar to scFOS, i.e., linear chains of fructose units linked by ~(2,1) fructosyl
fructose linkages, sometimes with a glucose endcap also linked by a ~(2,1) bond. Following
fermentation with microorganisms or hydrolysis the distinctions between them become less
noteworthy. Additionally, their activity and fate in the gastrointestinal tract is somewhat similar,

although not identical particularly for fructans of widely different DP. From a safety perspective,
both oligofructose from chicory or inulin and scFOS are compositionally and metabolically
similar. Indeed, all fructans contain molecules with DP of 3, 4, and 5, the components of scFOS,
usually in substantial quantities. Although detailed information on the DP distribution of fructans
is not always publicly available, in a GRAS notice (GRN 392) on oligofructose derived from
chicory that received a no question letter for addition of oligofructose to infant formula reported
percentages of the total oligosaccharide content provided by fractions of DP 3, 4, and 5 ranges
narrowly from 74.2 to 77.2%. This indicates that the infants in studies with oligofructose and
similar FOS were ingesting scFOS as much as 75% of their total oligosaccharide intake. Given
this the studies with oligofructose are applicable to the present GRAS assessment.
Yao et al. (2010) investigated the effects of infant formula containing oligofructose from
chicory at levels 0, 3, or 5 g/L on stool characteristics and composition. In this prospective,
randomized, double-blind, parallel-group study, 300 healthy formula-fed term infants aged 7-14
days were assigned to one of four, a-lactalbumin-enriched formulas for eight weeks: standard
term infant formula; formula with 40% of the palmitate in the sn-2 position; formula with high
sn-2 and 3.0 g oligofructose/L; or formula with high sn-2 and 5.0 g oligofructose/L. Additionally,
75 infants fed human milk served as a reference group. Tolerance was assessed via a parental
questionnaire and physician-reported study events. The primary outcome measure was mineral
content and stool soap at week 8; secondary outcome measures included stool characteristics and
GI tolerance.
In the study by Yao et al. (2010), 2 participants from the human-milk reference group, 2
from the high sn-2 group, 1 from the control group, 1 from the 3.0-g oligofructose group, and 0
from the 5.0 g oligofructose group withdrew. The infants receiving the high sn-2 formula,
whether with or without oligofructose had significantly less stool palmitate soaps and higher
bifidobacteria counts as compared to the control infants, resembling the human-milk reference
group. There was no difference in stool frequency. The high sn-2 group also had significantly
softer stools compared to the control infants, and the addition of oligofructose resulted in a
further dose-dependent increase in stool softness. The 5.0 g oligofructose group was not
significantly different from the human-milk reference infants. Similarly, the addition of
oligofructose significantly decreased stool calcium in a dose-dependent manner. Physician
reported GI events were few and were not different among the four formula groups and the
human-milk reference group; parental reports indicated no increase in the incidence of gassiness,
watery stools, or other symptoms of intolerance with the addition of oligofructose. The addition
of up to 5.0 g oligofructose/L to formula had no effect on growth (weight, length, head
circumference).
Lugonja et al. (2010) compared the bifidogenic effects of breast milk and prebiotic
supplemented infant formula. In this non-randomized, non-blinded, non-placebo-controlled trail,
21 healthy infants aged 5 to 16 weeks (mean= 8.6 weeks) were divided in to two groups. Group
one with 10 infants (7 boys and 3 girls) were breastfed, while other group with 11 infants (6 boys
and 5 girls) received formula containing 400 mg/100 ml of a blend of inulin and oligofructose
derived from chicory. Additional details of the fructans were not described. The relative
proportions of FOS and inulin in the blend was not reported, nor was the rationale for creating
the blend. During the trial duration of 28 days, daily measures of infants were taken for weight,
length, number of feeds, frequency of stooling, stool consistency (soft, normal, or hard), and any
indications of intolerance (loss of appetite, regurgitation, GI symptoms, and flatus). At baseline,

and on Days 14 and 28, stool samples were collected and analyzed for pH, organic acids, and
numbers of lactobacilli, total aerobes, total anaerobes, bifidobacteria, and fungi/yeasts. The
number of daily feeds was significantly higher in the breastfed group. Lactobacilli increased in
both groups while aerobes, anaerobes, and fungi and yeasts decreased, but there were no
significant differences between the formula and breastfed groups. The counts of bifidobacteria
increased significantly over the 28 days in both groups. Total organic acids increased and pH
decreased over time in both groups. Most stools from infants in both groups were of normal
consistency. The mean water content of the stools of infants receiving formula containing inulin
+ oligofructose was 77.9%, non-significantly lower than the mean water content of breastfed
infants' stools (81.2%). All infants grew at normal rates and there was no difference between the
groups. There were no significant differences between groups in measures of intolerance, stool
frequency, or stool consistency.
In another study, Kapiki et al. (2007) investigated the effect of a FOS supplemented
formula on gut flora of preterm infants. In this randomized, double-blind, placebo-controlled
study, 56 healthy bottle-fed preterm infants were enrolled. For this study, FOS was described as
having been produced by partial enzymatic hydrolysis of chicory inulin. All enrolled infants were
less than 14 days old (mean age= 7.0 ± 4/5 days), had gestational ages less than 36 weeks (mean
= 33. 7 ± 1.6 weeks), and had been admitted to a neonatal unit, but were otherwise healthy. Of the
56 infants, 24 received preterm formula with 400 mg maltodextrin (placebo)/100 ml formula,
while 41 infants received similar formula with 400 mg FOS/100 ml for 14 days. In this study, 9
infants failed to complete the study, 5 from the FOS group and 4 from the placebo group, for
reasons not related to the study. Over the full 14 days, infants in the placebo group gained
significantly more weight and had significantly greater arm circumference, while those in the
FOS group gained non-significantly greater length. Both formulas were well tolerated. The
intake of the FOS-supplemented formula produced a significantly higher frequency of defecation
and softer stools as well as significantly greater concentrations of fecal bifidobacteria and
bacteroides and significantly lower numbers of E. coli and enterococci. In the publication, it is
stated that "All infants tolerated well the two formulae," although the evidence supporting this
claim was not described. The investigators also stated, "We have documented that the addition of
a small quantity of FOS in the normal diet of preterm infants was well tolerated and resulted in a
rapid increase in the numbers of bifidobacteria and the proportion of infants colonized by
bifidobacteria."
In a randomized, double-blind study in infants, Hettler and Euler (2006) evaluated growth
and tolerance in infants fed formula supplemented with oligofructose (FOS) from chicory.
Healthy term infants were randomly assigned to 1 of 3 formulas (a bovine milk-based control
formula or identical experimental formulas supplemented with either 1.5 g/L or 3.0 g/L FOS) ad
lib for 12 weeks. Anthropometric measurements were recorded at baseline and at 4, 8, and 12
weeks. Adverse events and tolerance were recorded throughout the study, and blood samples
were drawn at baseline and at 12 weeks for a clinical chemistry panel. The study enrolled 297
infants, of whom 212 completed the trial. The infants were found to have grown appropriately.
All 3 formulas were judged to be safe and well tolerated based on growth, laboratory data, and
adverse event profiles. The high dose (3 .0 g/L FOS) group had less constipation than the other
study groups. The investigators concluded that Bovine milk-based term formula supplemented
with either 1.5 g/L or 3.0 g/L FOS is safe and supports normal growth.

Brunser et al. (2006) investigated the effect of probiotic or prebiotic supplemented milk
formulas on fecal microbiota composition of infants. In this randomized, double-blind trial, 116
healthy term infants were given a standard milk-based infant formula, the same formula with 200
mg/100 ml of oligofructose (from chicory), the same formula with 10 8 cfu L. johnsonii NCC533
(Lal)/g powder, or breast feeding, for a period of 13 weeks, followed by a 2-week washout with
standard formula. Parents maintained a record of formula intake and any adverse effects and
returned to the clinic every 15 days for health status evaluation and anthropometric
measurements. Seventy-six formula-fed infants completed the entire study; primary reasons for
withdrawal were failure to follow the protocol, antibiotic use, or illness. The investigators stated
that withdrawal rates did not differ across the three formula groups and none of the withdrawals
were associated with adverse reaction to the formula. All formulas were well tolerated and
average formula intake was similar for all three groups, resulting in an average intake of
oligofructose of 252 mg/kg bw/day. The number of adverse events per infant did not differ
between the three formula groups or between the formula-fed and breastfed infants, nor were
there any differences in growth measured by weight gain and length. The investigators concluded
that the study confirms a predominance of bifidobacteria in breastfed infants, and that the
concentration of oligofructose used in this study (200 mg/100 ml formula) was too small to have
a significant effect on the host microbiota.
In summary, the findings from the studies conducted with oligofructose or FOS derived
from other sources, such as chicory, shows that these ingredients are well tolerated in infants.
The findings from these studies suggest that scFOS, derived from sucrose and the subject of
present GRAS, at the intended use levels in infant formula is unlikely to cause adverse effects.
6.1.2.2. Studies in Children and Adults
Safety of scFOS has been described in several published clinical trials in children and
adult human subjects. These studies have been the subject of several comprehensive evaluations,
including several GRAS notices [GRN 44 (FDA, 2000), 537 (FDA, 2015), 605 (FDA, 2016a),
623 (FDA, 2016b), 717 (FDA, 2017), 797 (2018)] that have been reviewed by independent
expert panels and the FDA. Among these GRAS notices on scFOS, GRN 605 was submitted by
Tata. As the available information is extensively described in these previous GRAS notices,
including GRN 605 Tata, all these GRAS notices are incorporated in the present GRAS by
reference. The first GRAS notice on scFOS, GTC Nutrition (2000) established the ADI of 4.2
g/day scFOS for infant (<l year old). For the general population, scFOS ADI was established as
20 g/day. In these studies no serious adverse events of scFOS were reported. The available
information revealed only mild GI side-effects of scFOS consumption that included bloating,
abdominal discomfort, flatulence, and transient diarrhea. These GI effects are consistent with the
effects associated with intake of high levels of non-digestible fibers. Updated searches of the
recent scientific literature were conducted to identify any new studies relevant to the safety of
scFOS in children and adults. No recent studies on the effects of scFOS in adults or children
were located since the submission oflast GRAS notice in 2018 .
6.1.2.3. scFOS Studies in Piglets and Other Weaning Animals
The neonatal piglet is considered the best surrogate model to human infants with regards
to assessing the ability of test infant formula to support infant growth and development. The
available evidence indicate that neonatal piglet is similar in nutritional requirements, intestinal
physiology, and metabolism to the human infant. Additionally, the body composition of piglet is

similar to that of the premature human infant. Given this, the available studies of FOS in
neonatal piglets are described first followed by studies in other animal species.
In two experiments with neonatal pigs, Howard et al. (1995b) investigated the effects of
feeding scFOS on cecal and colonic microbiota, proliferation of cecal and colonic epithelial
mucosa, and short-chain fatty acid concentrations in the cecum. Although full description of test
article was not provided, the information described indicate that the product used in the study
appears to be scFOS. In the first experiment, male neonatal pigs (10/group) were fed diets
containing either O or 3 g scFOS/L of formula for 15 days and then the large intestine were
examined for changes in cecal and proximal colonic microbiota; cecal pH; short-chain fatty acid
concentrations; morphology of cecal, proximal, and distal colonic epithelial mucosa; gross
necropsy; and histopathology. Supplementation with scFOS did not alter cell counts of viable
bifidobacterial organisms or total anaerobic microbiota, cecal pH, or concentrations of short
chain fatty acids. Cecal mucosal cell density and labeled cells increased with FOS consumption.
Proximal colonic mucosa! crypt height, leading edge, labeled cells, proliferation zone, and
labeling index increased with scFOS consumption. Distal colonic mucosa! crypt height, leading
edge, cell density, labeling index, and labeled cells increased with FOS consumption. Gross
necropsy and histopathology found no significant lesions. In the second experiment, neonatal
pigs were fed diets containing either O or 3 g scFOS/L of formula for 6 days. Fecal samples were
collected on the first full day of feeding and on days 3 and 6 after initiation of feeding. On days 1
and 3, concentrations of bifidobacteria were similar between diets. However, on day 6, pigs
consuming FOS tended to have greater numbers of bifidobacteria. These data suggest dietary
consumption of FOS will enhance bifidobacteria populations and prevent colonic epithelial
mucosa atrophy in neonates fed an elemental diet.
In the first experiment by Howard et al. (1995b ), one pig receiving scFOS exhibited
intestinal lesions "suggestive of bacterial infection," and six pigs (5 receiving scFOS) showed
mild hepatocellular vacuolation. These hepatic changes were nonspecific and were attributed by
the pathologist to a variety of factors including hypoxia, stress, metabolic imbalance, and
anorexia. The investigators concluded that these effects were not significant. Of the 20 pigs, 16
showed pulmonary lesions of hemorrhage, congestion, or atelectasis, which were regarded as
acute lesions most likely due to handling during sample collection prior to sacrifice. The groups
assignments of the 16 pigs were not reported, but the authors reported that they "were not
associated with dietary factors" (Howard et al., 1995b). In this study, 36-hour-old piglets were
put on formula containing O or 3 g scFOS/L and no adverse effects were reported that were
attributed to the test article.
In another study, Tsukahara et al. (2003) investigated the effect of dietary scFOS
supplementation on luminal SCF A production and its influence on the morphometrical variables
of mucosa of the large intestine in six weaning piglets. After 7 days of adaptation, three pigs
were given a test diet containing scFOS (10%) ad libitum for 10 days. The other three remained
on the basal diet and served as controls. At the end of the experiment, the large intestines were
removed, and the cecum, gyri centripetales, gyri centrifugales, and rectum were separated. The
contents of each portion were collected and measured for SCFA concentration, pH, and moisture.
A micrometer was used to measure the crypt depth. The numbers of epithelial and mitotic cells
in the crypt columns were also counted. The concentration of SCF A was significantly higher in
piglets fed FOS than in the controls. The concentration of n-butyrate was markedly stimulated by
FOS. As compared to the control, the number of epithelial mitotic, and mucin-containing cells

was higher in piglets fed scFOS. Accordingly, the crypt depth was larger in the scFOS-fed
piglets. The luminal n-butyrate concentration showed a significantly positive correlation with the
crypt depth and the number of epithelial, mitotic, and mucin-containing cells. The investigators
concluded that "the beneficial roles of scFOS in the physiology of the large intestine rely on the
activity of intestinal microbiota."
In yet another study in piglets, Barnes et al. (2012) investigated the effects of partial
enteral nutrition, supplemented with the prebiotic scFOS in a neonatal intestinal failure piglet
model. In this study, male and female neonatal piglets (2 day old, n = 87) underwent placement
of a jugular catheter and an 80% jejunoileal resection and were randomized to one of the
following treatment groups: control (20% standard enteral nutrition/80% standard parenteral
nutrition PN), control plus prebiotic (10 g/L- scFOS), control plus probiotic (lx10 9 CFU
Lactobacillus rhamnosus GG [LGG]), or control plus symbiotics (scFOS + LGG). Animals (7-8
piglets/group) received infusions for 24 hours, 3 days, or 7 days, and markers of intestinal
adaptation were assessed. Prebiotic treatment increased ileal mucosa weight compared with all
other treatments and ileal protein compared with the control, regardless of day. Heal villus length
increased in the prebiotic and symbiotics group, regardless of day, specifically due to an increase
in epithelial proliferation. In the 7-day prebiotic group, peptide transport was upregulated in the
jejunum, whereas glutamine transport was increased in both the jejunum and colon. The
investigators concluded that scFOS prebiotic and/or symbiotics supplementation resulted in
enhanced structure and function throughout the residual intestine. No adverse effects were noted
from administration of 10 g scFOS/L in the parenteral formula, and the prebiotic was regarded as
"highly effective at inducing adaptation in the residual jejunum, ileum, and colon."
Correa-Matos et al. (2003) investigated the effects of fermentable nondigestible
carbohydrates in piglets infected with Salmonella typhimurium. In this study, 2-day-old
colostrum-fed piglets (12 piglets/treatment) were randomly assigned to receive saw's-milk
replacer formula alone (control) or control formula supplemented with 7.5 g/L of
methylcellulose, soy polysaccharides (soy fiber), or an undefined FOS for 14 days. The source
and composition of the supplements were not described. On day 7, half of the piglets in each
treatment group received an oral gavage of S. typhimurium 798 (originally isolated from a pig) or
saline. S. typhimurium infection produced diarrhea in the controls and in the methylcellulose
groups, but not in the soy polysaccharides or FOS groups. Heal lactase activity and physical
activity were significantly lower in the controls than in other groups after infection. Heal mucosal
barrier function was significantly impaired by S. typhimurium infection in the control and soy
polysaccharide groups, but was unaltered in the jejunum and colon. Overall, consumption of
FOS shortened recovery time and improved infection-associated symptoms in piglets infected
with S. typhimurium. The investigators concluded that, "because fermentable fiber enhances
intestinal function and reduces the severity of S. typhimurium infection-associated symptoms, it
may be a cost-effective way in which to reduce the severity of pathogenic infection-associated
symptoms in infants."
In another publication, Howard et al. ( 1995a) studied the effects of scFOS, XOS, and
gum Arabic on cecal and colonic microbiota in weaning rats and mice. In this study also two
experiments were conducted to determine if supplementing soluble fiber [FOS,
xylooligosaccharide (XOS) or gum arabic] to a semi-elemental diet would affect cecal and
colonic microbiota. Experiments 1 and 2 used identical dietary regimens; mice and rats were
given free access to a powdered semi-elemental diet. Animals were assigned to one of the four

following treatment groups: control, no supplemental dietary fiber, FOS, XOS and gum arabic.
Dietary fiber was supplied via drinking water at 30 g/L. In the first experiment, populations of
Bifidobacteria and total anaerobic flora were enumerated from the contents of the cecum and
colon of weanling mice. Consumption of FOS increased the concentrations of Bifidobacteria and
the ratio of Bifidobacteria to total anaerobic flora. In the second experiment, tissue from the
cecum and distal colon of weanling rats was examined for morphological changes of the mucosa.
Consumption of XOS increased cecal crypt depth and labeling index relative to the other three
treatments. Consumption of gum arabic and the control diet increased cecal proliferation zone.
Consumption of XOS and the control diet increased cecal cell density. Distal colonic crypt depth
was greatest in controls and rats fed FOS, intermediate in those fed gum arabic, and smallest in
those fed XOS. These results suggest that FOS effectively stimulates growth of Bifidobacteria
and XOS supports a modest enhancement of cecal epithelial cell proliferation.
Nakamura et al. (2004) investigated the effects of scFOS on the mucosa! immune system
in infancy using neonatal BALB/c mice. In this study, at 2 days of age, litter sizes were adjusted
to 4-6 pups and the pups and their dam were housed together and fed ad libitum diet containing 0
or 5% scFOS. Pups were weaned at 21 days of age and fed the same diets ad libitum to age 23,
30, 38, or 44 days. On days 28, 36, and 42, twenty-four-hour fecal samples were collected and
analyzed for IgA level. Following euthanasia, the small intestine and colon were removed,
luminal contents were flushed and analyzed for SCFA, segments were weighed, and the tissue
was homogenized and centrifuged for analysis of IgA. Feed intake and body weight did not
differ between the groups. Mice receiving scFOS had significantly higher levels of IgA in the
jejunum, ileum, and colon, as well as in the feces, and significantly higher levels of cecal acetate,
butyrate, and propionate. No adverse effects were observed.
Fukata et al. (1999) investigated the effects of competitive exclusion and ingestion of
scFOS on colonization of chicks with Salmonella enteriditis, in two separate experiments. Both
experiments used 1-day-old White Leghorn Hy-Line cockerel chicks caged. In both the
experiments, 60 chicks were divided into 4 groups (n=15): a control group; a competitive
exclusion group that received the control diet but was inoculated with an undefined bacterial
preparation; an scFOS group for which the feed was supplemented with 0.1 % scFOS; and a
combination-treatment group that received both interventions. In experiment 1, all chicks were
inoculated with S. enteriditis on day 7, while in experiment 2, chicks were inoculated on day 21.
Following inoculation, on day 1, week 1, and week 2, five birds from each group were
euthanized and their ceca evaluated for Salmonella spp., Escherichia coli, Lactobacillus spp.,
Bifidobacterium, and Bacteroides using plating techniques. In experiment 1, the enumeration of
S. enteriditis in the chicks inoculated with the competitive-exclusion preparation was
significantly decreased compared with the other three groups. In experiment 2, S. enteriditis was
significantly decreased in the scFOS group and the combination-treatment group. No significant
differences between groups were noted on cecal numbers of total bacteria, Bifidobacterium,
Bacteroides, Lactobacillus, or E. coli. The investigators concluded that low-dose feeding of
scFOS in the diet of chicks with a competitive-exclusion treatment is unlikely to a shift the
intestinal gut microbiota but may result in reduced susceptibility to Salmonella colonization. The
results of this study show that feeding of scFOS at 0.1% dietary concentration to 1-day-old
chicks for up to 35 days did not reveal adverse effects.
In summary, the available studies in weaning pigs, rats, mice and chicks indicate that
scFOS is unlikely to cause adverse effects. As the piglet is considered as a surrogate model for

human infants, studies conducted in these animals are applicable to the present GRAS
assessment. In the studies using piglet model, the exposure to scFOS was as follows: diet
containing scFOS ( 10%) ad libitum for 10 days; 3 g scFOS/L for 15 days, intestinal failure
model-10 g/L for 7 day; and 7.5 g/L in formula for 14 days. In these studies, no adverse effects
of scFOS were reported. Additional studies in chicks (0.1 % scFOS in diet), mice (water
containing 30 g scFOS/L for 14 days) and rats (water containing 30 g scFOS/L for 14 days) also
did not reveal adverse effects of scFOS. These findings from neonatal animal studies suggest that
proposed use of scFOS in infants by Tata is unlikely to cause adverse effects.
6.1.2.4. Specific Animal Toxicity Studies of scFOS
In an attempt to investigate safety and establish the no observed adverse effect level
(NOAEL) of scFSO, subject of present GRAS (FOSSEN CE™), Jain et al. (2019) conducted the
acute toxicity, 14-day dose range finding study, and subchronic (90-day) toxicity in Wistar rats.
6.1.2.4.1. Specific Acute Toxicity
In order to determine maximum tolerable dose (MTD), young adult healthy Wistar rats
(HsdHanTM) were administered a single oral dose of scFOS (dissolved in water) at dose levels
of 0, 2000, 5000, and 9000 mg/kg (n=5 rats/sex/group) (Jain et al., 2019). The rats were observed
for clinical signs or mortality, and body weights and feed consumption were measured. All the
rats were euthanized under isoflurane anesthesia on day 15, and gross pathological examinations
were performed. Oral gavage administration of scFOS to Wistar rats did not reveal any clinical
signs, mortality, on body weight changes, and feed consumption changes at 2000, 5000, and
9000 mg/kg bw. Necropsy at the end of study (day 15 post-dose) did not reveal any gross
pathological abnormalities. Based on these findings the MTD was considered to be more than
9000 mg/kg bw. The LDso of scFOS following oral administration to rats was more than 9000
mg/kgbw.
6.1.2.4.2. Specific Dose-Range Finding Study
For these investigations, young adult healthy Wistar rats (HsdHanTM) were orally
(gavage) administered scFOS at dose levels of 0, 2000, 5000, and 9000 mg/kg bw/day (n=5
rats/sex/group) for 14 consecutive days (Jain et al., 2019). The rats were observed for clinical
signs or mortality, and body weights and feed consumption were measured. On Day 15, all the
rats were euthanized under isoflurane anesthesia and blood samples were collected by retro
orbital puncture for clinical pathology (hematology, coagulation, and clinical chemistry), and
organ weights and gross pathological examination. Based on the increased cecum weight
observed in both sexes at all the doses tested, microscopic examination was performed on cecum,
colon, duodenum, jejunum, and ileum from all dose group animals.
No clinical signs or mortality were observed at the tested dose levels of 2000, 5000, and
9000 mg/kg bw/day. The body weights were unaffected at all the doses tested. A slight decrease
in feed consumption observed during treatment days 4-8 and 8-11 at the highest dose. However,
feed consumption during days 11-14 was comparable to the control group. Hence slight decrease
in feed consumption that was observed during initial days of the treatment was considered as
transient non-adverse finding. There were no scFOS related changes observed in hematology,
coagulation, and clinical chemistry parameters as well as in organs weight and gross pathology.
There were no scFOS related microscopic changes observed in cecum, colon, duodenum,
jejunum, and ileum of both the sexes. The findings from this study reveals that 14-day repeat

dose oral gavage administration of scFOS to Wistar rats did not cause any adverse toxicological
changes on the evaluated parameters at doses up to 9000 mg/kg bw/day.
6.1.2.4.3. Specific Subchronic Toxicity Study
The 90-day study was performed as per OECD guidelines for testing of chemicals (Test
Guideline No. 408, "Repeated Dose 90-Day Oral Toxicity Study in Rodents" adopted on
September 21, 1998) (Jain et al 2019). For these investigations, young adult healthy Wistar rats
(HsdHanTM) (n=lO rats/sex/group) were administered scFOS through oral gavage route at doses
of 0, 2000, 5000, and 9000 mg/kg bw/day for 90 consecutive days. In addition, two recovery
groups (n=5 rats/sex/group) such as control recovery and high dose recovery were included. All
standard parameters as per OECD guidelines were measured. These parameters included clinical
signs, body weights, feed consumption, ophthalmological examination, functional observation
battery, clinical pathology (hematology, clinical chemistry), urine analysis, necropsy, organ
weights, and histopathology.
There were no deaths, relevant clinical signs, or abnormal ophthalmological findings
noticed at any of the dose levels in this study (Jain et al., 2019). Few clinical signs and other
changes noted were considered incidental and not considered related to scFOS treatment. There
were no treatment related changes observed in neurological/functional examination carried out at
the end of treatment period for the main toxicity treatment groups and at the end of recovery
period for the toxicity recovery groups. Body weights were unaffected at 2000 and 5000 mg/kg
bw/day doses in males and at all the doses tested in females as compared to the control group.
The statistically significant lower body weights were observed on day 90 in animals treated at
9000 mg/kg bw/day when compared to the control group in males. The body weights were
slightly lower (without statistical significance) at 9000 mg/kg/day in both main and recovery
group males for the most part of the treatment period from week 7 till the end of the treatment
period and considered partially reversible at the end of the recovery period. The feed
consumption was unaffected at 2000 mg/kg bw/day (G2) in males and females as compared to
the control group. The statistical significant changes (decrease) in feed consumption were
observed at the doses of 5000 and 9000 mg/kg/day in males and females during the treatment
period and considered reversible during the recovery period (Jain et al., 2019).
No scFOS treatment related biologically significant adverse effects were noted in
hematological and coagulation parameters of both the sexes across the groups. There were few
statistically significant differences in hematology parameters in scFOS treated animals compared
to controls. These changes included decreased hemoglobin at 5000 mg/kg bw/day in males;
decreased mean corpuscular hemoglobin concentration at all FOS-treated groups in males and at
5000 and 9000 (main and recovery) mg/kg bw/day in females; increased mean platelet volume at
2000 and 5000 mg/kg/day in males and 9000 mg/kg bw/day recovery in males and females;
decreased absolute eosinophils at 9000 mg/kg bw/day in males; and decreased reticulocytes
(both absolute and%) at 9000 mg/kg bw/day recovery females. In the coagulation parameters,
decreased prothrombin time values at 9000 mg/kg bw/day recovery males were noted (Jain et al.,
2019). All the statistically significant changes observed were considered incidental and
toxicologically insignificant as the alterations were of minimal in magnitude and/or lacked the
dose progression and also microscopic correlation.
As regards changes in clinical chemistry parameters, no scFOS treatment related
biologically significant adverse effects were observed in of both the sexes across the groups.

There are occasional sporadic findings of statistically significant differences in the following
parameters from FOS-treated rats. These changes included decreased total cholesterol, total
proteins, and globulin at 5000 and 9000 mg/kg/day in males and at 9000 mg/kg/day in females;
alanine aminotransferase (ALT) at 9000 mg/kg/day recovery in males; decreased potassium at
9000 mg/kg/day recovery in males; and increased alkaline phosphatase at 9000 mg/kg/day in
females. There were no test item-related changes in the urinalysis parameters in treated rats as
compared to controls (Jain et al., 2019).
As regards organ weights, increase in absolute and relative cecum weight (with and
without content) was observed at 9000 mg/kg/day in both the sexes. However, this change was
not associated with any microscopic findings and hence considered as test item-related non
adverse effect. The cecum weight change was completely reversed in the recovery males,
whereas in females, it was partially recovered. Similar increase in cecum weight was also present
at 2000 and 5000 mg/kg/day in both the sexes and was attributed to test item administration. The
large doses of scFOS may result in cecal enlargement indicative of higher cecum weight, which
was considered to be a trophic effect and not a toxic effect. All other statistically significant
differences observed in organ weight and their ratios were considered incidental as the changes
were minimal in magnitude and/or lacked the microscopic correlation. There were no test item
related gross changes observed in male and female rats (Jain et al., 2019).
Histopathology findings from control and high dose (9000 mg/kg/day) groups did not
reveal any scFOS related microscopic changes observed in male and female rats at all the doses
tested. All the microscopic findings observed in males and females at 9000 mg/kg/day dose were
considered incidental/spontaneous and not related to test item administration, as they were
distributed randomly across the groups and/ or normally present in rats of this age. In addition,
observed microscopic findings were comparable to vehicle control group. Based on the findings
from this study, the oral gavage administration of scFOS (FOSSENCE™) at levels up to 9000
mg scFOS/kg bw/day is safe in Wistar rats without any adverse toxicological findings when
administered for 90 consecutive days. The no-observed adverse-effect level (NOAEL) can be
established as 9000 mg scFOS/kg bw/day (Jain et al., 2019). The findings from these specific
studies with the subject of present GRAS indicate that the proposed use of scFOS in infant
formula is unlikely to cause adverse effects.
6.1.2.5. Other Published Studies
6.1.2.5.1. Metabolism
The available published studies suggest that several non-digestible oligosaccharides and
polysaccharides act as prebiotic compounds. Among these, inulin, FOS and GOS are the most
commonly used in food. Pharmacokinetic studies of FOS demonstrate that it is not hydrolyzed
by human salivary or pancreatic enzymes and passes undigested and unabsorbed to the colon. In
colon, FOS is fermented by colonic microflora to short-chain fatty acids, carbon dioxide,
methane and hydrogen gases (Hidaka et al., 1986, Tomomatsu, 1994; Gibson and Roberfroid,
1995; Rumessen et al., 1990, 1998; Hess et al., 2011). The available studies in Wistar rats, as
well as in vitro studies, using pancreatic and small intestinal homogenates and purified sucrase
isomaltase complex, suggest that scFOS, like other fructans , is not hydrolyzed by the intestinal
enzymes but is fermented by gut microbiota (Oku et al. , 1984; Tsuji et al. , 1986; Tokunaga et al.,
1989; Bjork and Nilsson, 1991). The unfermented dietary FOS is excreted in the feces . The
kinetics of bacterial fermentation is inversely proportional to the degree of polymerization of the
Tata Chemicals Page 32 of63 FOS - IF-GRAS

fructan. The available evidence from studies in healthy human subjects (Stone-Dorshow and
Levitt, 1987; Rumessen et al., 1990; Molis et al., 1996; Alles et al., 1996; Rumessen and
Gudmand-Hoyerr, 1998; Castiglia-Delavaud et al., 1998; van Dokkum et al., 1999), as well as in
compromised adults with ileostomy (Bach Knudsen and Hessov, 1995; Ellegard et al., 1997)
suggest that nearly all ingested fructans, such as inulin, oligofructose, and scFOS reach the colon
where they are fermented by colonic bacteria.
Sivieri et al. (2014) investigated the prebiotic effect of FOS in the simulator of the human
intestinal microbial ecosystem (SHIME® model). The model was used to study the effect of
FOS on the fermentation pattern of the colon microbiota. Initially, an inoculum prepared from
human feces was introduced into the reactor vessel and stabilized over two weeks using a culture
medium. This stabilization period was followed by a 2-week control period during which the
microbiota was monitored. The microbiota was then subjected to a 4-week treatment period by
adding 5 g/day FOS to vessel one (the "stomach" compartment). A significant increase in the
Lactobacillus spp. and Bifidobacterium spp. populations during the treatment period was noted.
Overall microbial community was changed in the ascending colon compartment of the SHIME
reactor. FOS induced an increase of the SCF A concentration during the treatment period, mainly
due to significant increased levels of acetic and butyric acids. However, ammonium
concentrations increased during the same period. This study indicates the usefulness of in vitro
methods that simulate the colon region as part of research towards the improvement of human
health.
6.1.2.5.2. Toxicity Studies of FOS
In addition to above described specific toxicity studies with the subject of present GRAS
assessment, several studies of scFOS derived from sucrose have been described in the published
literature. The scFOS used in these studies appear to be substantially equivalent to the subject of
the present GRAS. These studies included acute oral toxicity studies in mice and rats, three
subacute studies, one subchronic study, one chronic study and two studies evaluating
developmental and maternal toxicity in rats. Additionally, in vitro mutagenicity and genotoxicity
studies in bacterial or mammalian cell models in the presence and absence of metabolic
activation have also been conducted with scFOS. In the repeat-dose toxicity studies, no
consistent treatment-related adverse effects of scFOS were noted and the NOAELs were the
highest doses tested. In these studies, scFOS related effects apparent at relatively high doses
included transient diarrhea, soft/watery stools, and intestinal weight increases. These effects are
well-established and consistent with the effects associated with intake of high-levels of non
digestible fibers and are considered to not be toxicologically relevant to humans. Decreases in
body weight in rats receiving high doses of scFOS are expected as a result of the decreased
caloric value of the diets rather than a direct toxic effect. In a 2-year study conducted with
Fischer 344 rats no evidence of carcinogenicity was reported and the NOAEL was determined to
be the highest dietary concentration tested of 5% (equivalent to 2170 and 2664 mg/kg bw/day for
males and females, respectively). No developmental or reproductive adverse effects were
associated with FOS consumption. Results of genotoxicity studies conducted with scFOS
consistently demonstrate the lack of a genotoxic effect in bacteria and mammalian cells in the
presence or absence of metabolic activation. These studies of scFOS are briefly described below.
6.1.2.5.3. Acute Toxicity Studies

In the acute oral toxicity studies, the effects of scFOS were tested in male and female
mice and Sprague Dawley rats. The available study details and findings from these studies are
summarized in Table 3. The results of these studies demonstrate that scFOS is of low acute oral
toxicity with median lethal dose (LDso) values exceeding 9000 mg/kg bw (highest dose tested) in
both mice and rats.
Table 3. Acute Toxicity Studies of scFOS in mice and rats
Description of Dose&
Test Species Test Article Duration Findings Reference
tMale and female scFOS Single gavage No deaths occurred and there were no Takeda and
UcL-IcR mice (6 doses of 0, 3, differences in body weight gain between Niizato
imice/sex/dose) 6, or 9 g the test and the control animals. No (1982);
scFOS/ abnormalities were seen in either sex. mouse study
kgbw The LDso for oral administration of
scFOS to rats in this study was > 9000
mg/kgbw.
!Male and female scFOS Single gavage There were no deaths and no Takeda and
/Sprague Dawley doses of 0, 3, abnormalities or changes in body weight Niizato
!rats (6 rats/sex/ 6, or 9 g of animals of either sex. The LDS0 for (1982); rat
ldose) scFOS/ oral administration of scFOS to rats in study
kgbw this study was> 9000 mg/kg bw.
6.1.2.5.4. Repeat-Dose Toxicity Studies

A summary of short-term, subchronic and chronic toxicity studies of scFOS is provided
in Table 4. The findings from the available published toxicological studies suggest that various
scFOS preparations are of low oral toxicity in repeat dose studies in rodents. These published
and commonly available toxicity studies of scFOS have been the subject of several critical and
independent evaluations by regulatory and other agencies. These repeat-dose published toxicity
studies did not reveal any toxicologically significant effects of relevance to humans following
oral administration of scFOS. In these studies, NOAEL determinations have been :consistently
reported as the highest doses tested.
In a review article, Carabin and Flamm (1999) described the findings from subacute
studies that were conducted by Takeda and Niizato (I 982). The findings from these 6-week
gavage and feeding studies of scFOS in Wistar rats support NOAELs of 4500 to 5000 mg/kg
bw/day (highest doses tested). Tokunaga et al. .(1986) reported that male Wistar rats consuming
FOS at dietary concentrations of 10 and 20% (equivalent to approximately 4185 and 7795 mg/kg
bw/day, respectively) experienced transient watery stools during the first few days of
administration and increased small and large intestine weights, and increased fecal and decrea,sed
gastrointestinal transit time when in the diet for 6 to 8 weeks. Meiji Seika Kaisha (1982) reported
dose-related increase in diarrhea, soft stools, cecal distension, and intestine weights for rats fed
up to 20400 mg/kg bw/day for 90 days. Additional details of the study were not reported.
In a subchronic toxicity study. (Boyle et al., 2008), Sprague-Dawley rats were fed
standard· rodent chow for 13 weeks with 0, 0.55, 1.65, 4.96~ or 9.91 % oligofructose, replacing
cornstarch. In this study, there were no reports of treatment-related adverse effects in terms of
food intake, body weight, body weight gain, clinical chemistry, hematology, clinical
Tata Chemicals Page34 of63 FOS- IF-GRAS

observations, or histopathology even at the highest dose tested. The NOAEL was the highest
dose tested (4680 mg oligofructose/kg bw/day).
In a chronic feeding study, Clevenger et al. (1988) investigated the toxicity and
carcinogenicity of FOS in rats following exposure for 104-weeks (Table 4). In this study, male
and female Fischer 344 rats (12-13/sex/dose) were fed diets containing scFOS at levels of 0, 0.8,
2.0, and 5.0% (equivalent to 0, 341, 854, and 2,170 mg/kg bw/day for male rats and 0, 419, 1,045,
and 2,664 mg/kg bw/day for female rats) for two years. All standard parameters for such studies
were studied. In all groups, some mortality was observed; however, it was not considered
treatment-related. Exposure to scFOS did not affect feed intake, body weight gain, feed
conversion efficiency, absolute organ weights, or any hematology outcomes. A slight elevations
in sodium and chloride in male rats was noted. In male rats in the mid-dose group, exposure to
scFOS showed slightly elevated levels of blood glucose and creatinine, but the creatinine levels
in males in the high-dose group decreased. Other outcomes did not significantly differ between
test groups and the controls. In female rats, except for a slight elevation of uric acid in the low
and mid-dose groups, all blood chemistry parameters were similar to those of the controls. No
test-article-related macro- or microscopic changes were found in either males or females. The
NOAEL was established as 5%, the highest concentration tested, equivalent to 2170 mg/kg
bw/day for males and 2664 mg/kg bw/day for females.
As regards the carcinogenicity-related observations, Clevenger et al. (1988) reported
similar numbers of neoplastic lesions (e.g., pheochromocytomas, thyroid C-cell adenomas,
leukemias, and pituitary adenomas) in the scFOS-treated animals and controls, with the
exception of pituitary adenomas. In male rats, the incidence of pituitary adenomas for the 0, 0.8,
2.0 and 5.0% dose groups was 20, 26, 38, and 44%, respectively. The historic incidence of
pituitary adenomas in F-344 male rats from the test laboratory ranges from 1 to 49%. While the
incidence of this tumor was well within historical range for all male rats, the incidence in the two
highest dose groups (2.0 and 5.0%) was significantly greater than the incidence in the controls.
In the female rats, a negative trend in the incidence of pituitary adenomas was recorded. The
significance of a dose-related trend was equivocal in that one trend test showed a significant
trend, whereas another test did not. If males are compared to females, a similar but opposite
dose-response trend is noted. This dichotomy has no apparent biological basis. If male and
female pituitary adenoma incidences are combined, no significant across-dose group difference
are found. All of these observations point toward the conclusion that the higher incidence of
pituitary adenomas in FOS-treated male rats is a chance artifact. Such chance artifacts can arise
when large numbers of statistical comparisons are made. In this study, 54 comparisons were
made, and 1 - 3 significant results would be expected by chance alone at the significance levels
of 0.01 and 0.05, respectively. These observations suggest that higher incidence of pituitary
adenomas in males was not treatment related. The findings from this study indicate that FOS is
not carcinogenic.
Tata Chemicals Page 35 of 63 FOS- IF-GRAS

Table 4. Summar'' of Short-term, Subchronic and Chronic Toxicity Studies of scFOS Conducted in Rats
Species strain Route and NOAEL
(NoJsex/group; Dose(mg/kg Duration (mg/kg Other Observations Reference
age/weieht) bw/dav) bw/dav)
Short-term Toxicit ~ Studies
! Body weight in 10,000 group
t Cecum and colon weights in
both treatment groups
i Small intestine weights in
Dietary: 0 10,000 group
Wistarrats (control),
6-8
i Fecal weight and ! GI transit Tokunaga et
(6M/group; 40- -4185, ~779SC NR time in both treatment groups
weeks al. (1986)
50g) scFOS ! Serum triacylglycerol and t
Neosugar® fecal excreted neutral sterols and
volatile fatty acids
During the first few days FOS
administration transient watery
stools
No mortalities or abnonnalities
Minor t body weight in 3000 and
4500 groups (stat. sig. not
reported)
Takeda and
Gavage: 0 No consistent, treatment-related
Niizato
(control), findings in serum chemistry
Wistar SPF rats (1982);
1500, 3000 or parameters (occasional
(ISM/group; 6-7 6 weeks 4500 summarized
4500 fluctuations reaching statistical
weeks old) in Carabin
scFOS (DPav significance were considered
and Flamm
=3.5) spurious - further details not
(1999)
reported)
Swollen appendix in rats
receiving treatment
(number/~ oup not reoorted)
No mortalities or treatment-
related abnormalities
Diarrhea reported on the 10th day
ofFOS administration (no
additional details reported)
! body weight in FOS treated Takeda and
Gavage:0 animals [(week 1-5)- stat sig. Niizato
Wistar SOP rats (control), not reported)], normalized near (1982);
(18M/group; 6-7 ~2500,~5000 8 6weeks 50Q0b completion of study summarized
weeks old) scFOS(DPav FOS related J, in cholesterol (stat. in Carabin
=3.5) sig. not reported) and Flamm
Swollen appendices were (1999)
reported at Week 2 and Week 6
necropsies (number/group not
reported)
No treatment related toxicity
comoared to controls
Subchronic Toxicitv Studv
No significant changes in clinical Meiji Seika
Dietary: Up to
chemistry, hematological or urine Kaisha
Rats (strain. 20,400
parameters and no abnormalities (1982),
number, sex, age scFOS (no 90 days NR
upon gross or histopathological cited in
not identified) further details
examination GRN44
reported)
Dose related t in diarrhea, soft (GTC
Tata Chemicals Page36of63 FOS- IF-GRAS

stools, cecal distension, intestine Nutrition,
weights 2000)
Chronic Toxicity and Carcinoi?enicity Study
Dietary:
Male: 0, 341,
854, and 2170 No dose-related effects on
2170
Fischer 344 rats Female: 0, survival, growth, hematological
104 (malesl Clevenger
(SO/sex/group; 4 419, 1045, and or clinical chemistry parameters,
weeks 2664 et al. (1988)
weeks old) 2664 organ weights or neoplastic
(females)h
scFOS lesions
Neosugar®
ffiP=2-4)
j = Increase; ! =decrease; DP = degree of polymerization; DPav = average degree of polymerization; GI =
gastrointestinal; GRN = GRAS registration notification; F = female; FOS = fructo-oligosaccharide; M = male;
NOAEL = no observed adverse effect level; NR = not reported; • Calculated using U.S. FDA, 1993; bStudy
authors did not provide a NOAEL, values were derived based on reported study findings; "Calculated using the
food intake values presented in the study report and weight of rats from U.S. FDA, 1993
6.1.2.5.S. Developmental and Reproductive Toxicity Studies

In addition to above described toxicity studies, the findings of a developmental toxicity
study in rats were also summarized Carabin and Flamm (1999) from an unpublished study by
Henquin (1988). The available study related details are provided in Table 5. In this study, dietary
exposure of scFOS at concentrations up to 20% (equivalent to approximately I 0000 mg/kg
bw/day) did not result in developmental toxicity. In the study summary, fetal markers other than
body weight were not further described. During the nursing period, 'a growth delay was
observed for the pups (specifically males) in the test group,' which was attributed to the
restricted nutritional status of the lactating mothers (who were consuming a diet with an
essentially non-caloric content of 20%, far above recommended levels to avoid nutritional
disturbances). The reviewers concluded that 'a diet containing 20% FOS has no significant
effects on the course of pregnancy in rats and on the development of their fetuses and newborns.'
In another study by Sleet and Brightwell (1990) also summarized in Carabin and Flamm
(1999), maternal and developmental toxicity of FOS at dietary concentrations up to 20%
(equivalent to approximately 10,000 mg/kg bw/day) were investigated. In this study, rats during
postcoitum days Oto 15 were fed a diet containing FOS. No treatment related adverse effects
(diarrhea), or differences in pregnancy outcome or in utero development were noted.
Route and Dose Duration Other Observations

Reference
No treatment effect on number of Henquin (1988);
pregnancies or fetus or newborn weights; described in
Dietary: 0 or
J.. Body weight during nursing period was Carahinand
10,000mg
Wistar rats {29F; reported in the treated pregnant rats and Flamm (1999)
scFOS/kg Gestation
n=l2 treatment pups; Diarrhea was observed in treated
bw/day(no days 1-21
and n= 17 control) pregnant rats (number not reported) during
further details
the first week and soft stools in weeks 2
reported)
and 3 for this group; Growth delay in male
u s in test ou 3

No treatment related adverse events; No Sleet and
deaths or diarrhea reported; ! Body weight Brightwell (1990);
Dietary: 0 or
237Samg on postcoitum Day 2 in all FOS treated described in
rats compared to control; Dose related Carabin and
scFOS/kg
Flamm ( 1999)
bw/day (Day O- decrease in body weight for FOS treated
6 postcoitum) rats; Body weight and body weight
Sprague Dawley changes in 2,500 and 5,000 mg/kg bw/day
(CrL CD (SD) Dietary: 0, 2500, groups were similar among groups from
Days 0-15
BR) rats 5000, or 10,0003 Day 12-15
postcoitum
Pregnant female mgscFOS/kg No remarkable findings at necropsy; No
rats (24-27/group) bw/day (Day 6 - treatment related effects on number of
15 postcoitum) pups/litter, the sex ratio, and viability of
both the embryo and the fetus or structural
scFOS (no development of fetuses; t Fetal weights of
further details 10,000 mg/kg bw groups compared to
reported) control, no other reduction in litter or fetal
weights
t =Increase;!= decrease; a calculated using U.S. FDA, 1993
6.1.2.5.6. Mutagenicity and Genotoxicity Studies

Clevenger et al. (1988) investigated the genotoxic potential of commercially available
scFOS (Neosugar®). These assays included, microbial reverse mutation assays (Ames assay) in
Salmonella typhimurium and Escherichia coli WP2 uvrA, mammalian cell mutation assay with
mouse lymphoma L5178Y cells; and induction of unscheduled DNA synthesis (UDS) in human
epithelioid cells (HeLa S3). The reverse mutation and unscheduled DNA repair assays were
conducted in accordance with the OECD guidelines and the mammalian cell mutation assay
conducted according to recognized methods. The findings from these assays are provided in
Table 6. The results of these studies suggest that scFOS is not genotoxic in bacteria and
mammalian cells in the presence or absence of metabolic activation.
Table 6. In vitro Genotoxicitv Studies on scFOS

Metabolic Reference
Test system Concentration Result
Activation
Bacterial reverse mutation assay
(Salmonella typhimurium T A98,
0, 50, 150, 500, 1500,
TAlO0, TA1535, TA1537, and ±S9* Negative Clevenger et al., 1988
or 5000 µg/plate
TA 1538 and Escherichia coli
WP2uvrA
Mammalian cell mutation assay 2000, 3000, 4000 or
± S9* Negative Clevenger et al., 1988
(mouse lym phoma L5178Y cells) 5000 u!!'./ml
25,50, 100,200,400,
Unscheduled DNA synthesis
800, 1600, 3200,
[Human epithelioid cells (HeLa
6400, 12,800, 25,600, - Negative Clevenger et al., 1988
S3)]
51,200 uwml
*S9= Metabolic activation with Aroclor 1254-induced rat liver S9; scFOS - short chain fructo-oligosaccharides

6.1.3. Available Corroborative Safety Evidence
6.1.3.1. Regulatory Agency Review
Based on FDA GRAS Notices inventory website, the FDA has received six GRAS
notices on FOS preparations, all of which have received GRAS status for use of FOS as a food
ingredient in a variety of conventional foods, including infant formula (GRN 44, 537, 605, 623,
717 and 797) (FDA, 2000; 2015; 2016a; 2016b; 2017; 2018). The currently marketed FOS
products are derived from sucrose is enzymatically converted to FOS following action of ~
fructofuranosidase obtained from different non-toxigenic and non-pathogenic strains of
microorganisms. Given the use of similar manufacturing processes, the differences between
various FOS products would be limited to minor variations in the compositional distribution of
the glucose-fructose disaccharides (FOS), and to differences in the residual levels of other sugars.
This also suggests that the safety information on FOS products can be interchangeably used.
The safety related information of scFOS have been extensively described in the 2014
FDA GRAS notification (GRN, 537) on scFOS for its uses in infant formula by Ingredion (2014).
In a subsequent GRAS notification (GRN, 717), Galam (2017) has also described available
safety information on uses of scFOS in conventional foods (Galam, 2017). In addition to these
two GRAS notices, in four additional GRAS notices (GRN, 979; GRN 623; GRN, 605; and
GRN, 44) the safety data on scFOS has been summarized. Among these GRAS notices on the
use of scFOS, GRN 605 was submitted by Tata. The comparative data and information from all
these GRAS notices is provided in Table 7. This comparison of these GRAS notices suggest that
all these products are similar. Furthermore, there is one more GRAS notice on oligofructose
(GRN, 392) that also describes the safety related information on FOS derived from chicory. FDA
did not question the acceptability and suitability of the available evidence to support the
proposed uses described in these seven GRAS notices, including its uses in infant formula, and
replied to all these notifications, including the GRAS notice by Tata, that the agency had 'no
questions' regarding the conclusions that the scFOS or oligofructose is GRAS for the intended
uses. Tata is hereby incorporating all the toxicology and human tolerance studies discussed in
these previous GRAS notices by reference (NFBC, 2018; Galam, 2017; NFBC, 2016; Tata,
2015; Ingredion, 2014; Pfizer, 2011; GTC, 2000).
As can be noted from the comparison given in Table 7, the subject of present GRAS
assessment, scFOS for use in infant formula is substantially equivalent in its specification and
composition (including disaccharide polymers as well as degree of polymerization) to that of
previous GRAS notices, GRN 797 and GRN 537, for uses of scFOS in infant formula.
Additionally, in these three GRAS notices, scFOS is proposed for uses in infant formula at same
use levels. In these three GRAS notices, the enzyme, ~-fructofuranosidase, derived from
microorganism, is used in the manufacturing of FOS . The levels of scFOS (95%) in these three
GRAS notices is same. The individual scFOS molecules such as 1-kestose (GF2), Nystose (GF3),
and Fructofuranosylnystose (GF4), as well as residual levels of sugars is very similar.

Table 7. Comparison of the Subject of Present GRAS, scFOS, with other FDA Accepted GRAS Notices
Current GRN GRN
Constituents GRN717 GRN623 GRN605 GRN44
GRAS* 797* 537*
Sucrose + Sucrose+ Sucrose+ Sucrose+ Sucrose + Sucrose+ Sucrose+
Manufacturing fungal fungal fungal fungal fungal fungal fungal
enzyme enzyme enzyme enzyme enzvme enzvme enzyme
Total FOS (%) 2:95 2:95 NLT95 95±2 2:95 95±2 >95
1-kestose (GF2)
NLT30 NLT30 NLT30 NLT30 NLT30 35-43** 35±6
(%)
Nystose (GF3)
NLT45 NLT40 NLT45 NLT40 NLT40 42-48** 50±6
(%)
Fructofuranosylny
NLT5 NLT5 NLT5 NLT5 NLT5 6-11 ** 10±4
stose (GF4) {%)
Sugars(%) ~5 ~5 NMT5 5±2 ~5 5±2% <5
Term Term
Term Infant Multiple Multiple Multiple Multiple
Intended uses Infant Infant
Formula foods foods foods foods
Formula Formula
4g/L
4g/L
4 g/L starter starter
starter
formula; 5 formula; 5 0.4to 0.4to 0.4 to 0.4to
Use levels formula; 5
g/L follow g/L follow 6.7% 6.7% 6.7% 6.7%
g/L follow
on formula on
on formula
formula
828 828 828
9.09 g/day 12.8 g/day 12.8 g/day 12.8 g/day
EDI mg/kg/day mg/kg/day mg/kg/day
(90 th %) (90th %) (90th %) (90th %)
(90th %) (90th %) (90th %)
At At
At proposed proposed proposed
ADI use levels use levels use levels 20 g/day 20 g/day 20 g/day 20 g/day
(4 or 5 g/L) (4or5 (4 ors
g/L) wL)
Safety Totality of Totality of Totality of Totality of Totality of Totality of Totality of
determination evidence evidence evidence evidence evidence evidence evidence
*The three shaded columns are for GRAS notices for use of scFOS in Infant Formula, while the other GRAS
notices are for use of scFOS in conventional foods. **Based on ranges from batches given in the GRAS notice.
Given the structural and chemical similarity of scFOS preparations that have been
concluded GRAS (e.g., GRN 797 and 537) by NFBC (2018) and Ingredion (2014) with the
current GRAS (Table 7), a discussion of publicly available data and information relevant to the
safety of scFOS is incorporated by reference to studies described in GRN 797 and 537.
Additionally, in GRN 797 some safety related and other question raised by FDA for GRN 537
are discussed. Based on all available information, there exists no evidence in the available
information on scFOS that demonstrates, or suggests reasonable grounds to suspect, a hazard to
infants when scFOS is added as a prebiotic ingredient to non-exempt infant formula at levels up
to 400 mg/100 ml in starter formula as consumed and 500 mg/100 ml in follow-on formula as
consumed. Additionally, given the similarity between the GRAS notices (GRN 797 and GRN
537) and the subject of this present GRAS assessment, as well as other information available,
Tata has concluded that the scFOS it intends to market for uses in infant formula is safe and
GRAS.
The Foods Standards Australia New Zealand (FSANZ) has evaluated the safety of FOS
for its uses as an alternative to inulin (FSANZ, 2013). Based on published information

characterizing the metabolism of FOS, published studies characterizing the toxicity of FOS in
animal models and published studies evaluating the safety and tolerance of FOS in humans
(children and infants), FSANZ concluded that FOS is technologically suited to its proposed use
and complies with international specifications. FSANZ noted that no adverse effects on growth,
hydration status, nutrient intake, frequency and nature of adverse events, gastrointestinal
intolerance, stool consistency and frequency, or fecal flora were observed in studies conducted in
healthy infants or young children at amounts of FOS up to 3.0 g/L for periods ranging from 1
week to approximately 3 months.
In addition to above described information on FOS, several other structurally related P2-1
fructan preparations also have received GRAS status for use as food ingredients (e.g., GRN 118,
392, 477 and 576- all these GRAS notices and FDA responses to these GRAS assessments are
available at FDA GRAS Inventory webpage). Although the related inulin type fructans have
similar chemical composition to scFOS and are expected to have a similar toxicological and
physiological profile following ingestion, these oligomers typically display a higher molecular
weight distribution.
6.1.3.2. Unpublished Corroborative Studies
In GRN 537 (Ingredion, 2014) on scFOS and also in FSANZ (2013) on FOS, two
unpublished on the effects of scFOS in infants are summarized. These publicly available
unpublished studies with scFOS are summarized in Table 8.
In the first unpublished randomized, double-blind, placebo-controlled trial, 63 healthy
term infants (age 4-10 weeks) were fed control formula for 2 weeks, followed by a whey
enriched formula containing 0, 150, or 310 mg scFOS/100 ml for additional two weeks. The
groups as enrolled included 21, 22, and 20 infants, respectively, with a mean age of 43±4 days.
Formula intake, growth, stool characteristics, and tolerance were assessed on days 1, 15, and 29.
Urine was collected on days 15 and 29 and blood was collected on day 29 for analysis. One
infant from the control group, 5 from the low-scFOS group, and 4 from the high-scFOS group
failed to complete the study. Withdrawal of one infant from control-group, two from the low
scFOS infants, and three from the high-FOS infants was due to intolerance, while the remainder
was attributed to protocol failures. Intolerance withdrawals were based on vomiting or spit-up,
diarrhea or watery stools, fussiness, increased stool frequency, or weight loss; there were no
differences in reported adverse events among feeding groups.
In this first study (lngredion, 2014; FSANZ, 2013), no significant differences among
groups were reported in formula intake, growth, stooling patterns, tolerance, or in any of the
outcomes measured in blood or urine. The blood analysis did not reveal the presence of kestose
or nystose, but these molecules were found in the urine of most of the infants who received
scFOS-containing formula for 2 weeks (GF2 in 55% and GF3 in 64%). The only statistically
significant difference in the microbiota was a reduction in Clostridium spp., in infants receiving
scFOS as compared to the control group. The investigators concluded that "Infant formulas
containing added FOS at the levels provided ... are well tolerated and support normal growth in
term infants." No explanation for the appearance of scFOS residues in urine but not in blood was
provided. However, the levels found in urine exceeded the detection limits by only small
amounts and, although the analytical methods and limits of detection in the blood analyses were
not described, it may be that these limits were higher for the blood analyses than for those in
urine and scFOS residue levels simply failed to reach detection limits.

In the second unpublished randomized, double-blind, placebo-controlled, multicenter trial
(Abbott 1993; FSANZ; 2013; Ingredion, 2014), 102 healthy term infants aged 1-8 days were
randomized to receive formula containing O (n = 52) or 300 (n = 50) mg scFOS/100 ml formula
for approximately 16 weeks (to 112 days of age). Additional group of 25 healthy breast-fed
infants aged 0-9 days served as human-milk reference group. Of the 70 infants receiving formula,
34 infants that were fed formula without scFOS and 36 consuming scFOS-containing formula, as
well as 23 of the 25 fed human milk.-fed infants completed the study. Protocol errors were
responsible for the loss of 12, 6, and 2 infants from the non-scFOS formula group, the scFOS
formula group, and the human milk group, respectively. Six infants were withdrawn from the
non-scFOS formula group and 8 from the scFOS group due to adverse events: symptoms of milk
intolerance (2 and 4 infants, respectively), diarrhea or watery stools (2 and 1 infants),
constipation (2 and 1 infants), and colic or gassiness (1 scFOS-group infant each). Differences
among groups were not statistically significant.
In the second study, there were no differences among groups in measures of weight,
length, or head circumference at any time during the study, nor did the formula groups differ in
feeding frequency or intake, feedings with spit-up or vomit, stool frequency, or stool consistency,
although the human milk-fed infants had significantly softer and more frequent stools than the 2
formula groups. Levels of total cholesterol in blood were significantly higher in the human milk
group than in either formula group, but levels of AST and ALT were similar in all groups. No
blood samples from any infant had detectible scFOS trimers or tetramers, but they were
consistently found in urine from infants receiving formula containing scFOS. No urine sample
contained detectible ketones. The investigators concluded that "infant formulas containing added
FOS at .. . up to 3 g/L are well tolerated and support normal growth in term infants. The addition
of the fermentable fiber at these levels, however, has only small effects on fecal microflora."
' Inf:ants *
T able 8. UDPUblis bed stlld.1es WI'th SCFOS m
Dose, Study Design,
Reference Duration Objective Subjects Results
Six infants were withdrawn from the non-scFOS
formula group and 8 from the scFOS group due to
adverse events: symptoms of milk intolerance (2
102 and 4 infants, respectively), diarrhea or watery
healthy stools (2 and 1 infants), constipation (2 and 1
Randomized, term infants), and colic or gassiness (1 scFOS-group
double-blind, infants infant each). Differences between groups were not
placebo- aged 1-8 statistically significant. There were no differences
scFOS O or3 controlled, days(and between groups in measures of weight, length, head
Abbott g/Lformula multicenter 25 healthy circumference, feeding frequency or intake,
(1993) for about 16 study of the breast-fed feedings with spit-up or vomit, stool frequency, or
weeks (to 112 safety and infants stool consistency, although the human- milk-fed
days ofage) bifidogenic aged 0-9 infants had significantly softer and more frequent
effect of days as a stools than the 2 formula groups. Levels of AST
scFOS in human- and ALT were similar in all groups. No blood
infant formula milk samples from any infant had detectible scFOS
reference trimers or tetramers. No urine sample contained
group) detectible ketones.
No differences were seen between the groups in
populations of Bifidobacteria, Bacteroides, or
Clostridia spp., or C. difficile, but counts of

Lactobacillus spp. were significantly higher among
infants receiving the scFOS-supplemented formula.
The authors concluded that "infant formulas
containing added FOS at ... up to 3 g/L are well
tolerated and support normal growth in term
infants."
One infant from the control group, 5 from the low-
scFOS group, and 4 from the high-scFOS group
failed to complete the study; withdrawal of the
single control- group infant, 2 of the low-scFOS
infants, and 3 of the high-FOS infants was due to
intolerance, while the remainder were attributed to
protocol failures.
Randomized,
63 healthy Intolerance withdrawals were based on vomiting or
double-blind,
term spit- up, diarrhea or watery stools, fussiness,
placebo-
scFOS 0, 1.5, infants increased stool frequency, or weight loss; there
controlled
Abbott or aged 4-10 were no differences in reported adverse events
study of the
(1992) 3.1 g/L weeks among feeding groups.
safety and
formula for 2 with a No significant differences among groups were
bifidogenic
weeks mean age reported in formula intake, growth, stooling
effect of
of43±4 patterns, tolerance, or in any of the outcomes
scFOS in
days measured in blood or urine. No kestose or nystose
infant formula
was detected in the blood of any infant. Infants
receiving scFOS had significantly reduced
Clostridia spp. as compared to the control group.
The authors concluded that "Infant formulas
containing added FOS at the levels provided ... are
well tolerated and support normal growth in term
infants."
*Adapted from GRN537*
6.1.3.3. Natural Occurrence

Oligosaccharides, including FOS, occur naturally in plants and are commonly consumed
by humans in foods. FOS occurs in a number of plants such as onions, Jerusalem artichokes,
bananas, lettuce, asparagus, rye, garlic and wheat (rough and bran forms) (GTC Nutrition, 2000;
Bernet et al., 2002). Some grains and cereals, such as wheat and barley, also contain FOS
(Campbell et al., 1997). The Jerusalem artichoke and its relative yacon 1 together with the Blue
Agave plant have been reported to contain the highest concentrations of FOS of cultured plants.
Campbell et al. (1997a) extensively analyzed and characterized the naturally occurring FOS
levels in a variety of plants. Of the 25 samples analyzed for FOS content, 20 showed detectable
levels of FOS . In these samples, the FOS content ranged from 0.1-0.2 mg/g for most (12/20) of
the fruits . The highest FOS content was found in ripe bananas, which contained 2.0 mg/g FOS.
Of the 40 vegetable samples analyzed, 16 did not contain FOS. An additional 6 vegetables
contained 0.1 or 0.2 mg/g FOS, while the remaining 16 vegetables contained from 0.3 to 58.4
mg/gFOS.
The available information suggests that humans consume FOS on a daily basis following
ingestion of plants that naturally contain FOS. An estimate of FOS intake from commonly
1 The yac6n is a species of perennial daisy traditionally grown in the northern and central Andes from Colombia to
northern Argentina for its crisp, sweet-tasting, tuberous roots.

consumed plants was provided in GRN 44 (GTC Nutrition, 2000). For this analysis, data
provided by Campbell et al. ( 1997) for the content of FOS was used along with food intake data
available for the U.S. population from the 1994-96 United States Department of Agriculture's
(USDA) Continuing Survey of Food Intakes by Individuals (CSFII). Based on the foods included
in the analysis reported by Campbell et al. (1997), the mean FOS intake for adults in the U.S.
was estimated as 114 mg/day. For adults, an upper bound estimate of daily FOS intake, based on
the 90 th percentile food intake was determined as 248 mg/day. The food types that contributed
the most to FOS consumption were onions, bananas, lettuce, and wheat (in rough and bran
forms).
6.1.3.4. Current Uses
FOS and other prebiotic ingredients are increasingly being recognized as useful dietary
tools for the modulation of the colonic microflora toward a healthy balance. FOS represents only
a fraction of the inulin class of carbohydrates known as fructans. This class includes different
chain length polymers such as inulin, oligofructose and FOS. Thus, inulin is a composite
oligosaccharide that contains several FOS molecules. These polymers are chemically similar
entities and share the same basic structure of p (2-1) linked fructosyl units, sometimes ending
with a glucosyl unit. As all these fractions are mixtures of molecules that differ only in chain
length, they can be described by their range and average degree of polymerization. Various terms
describing fructans have been used interchangeably in the published literature. Currently, there
are several commercial sources of FOS, inulin, and oligofructose. These products are sold and
consumed as fat replacements and sugar substitutes for use in a variety of foods such dairy
products, candies and chocolates, spreads, baked goods and breakfast cereals, meat products, ice
cream and frozen yogurt (GTC Nutrition, 2000). In the U.S., FOS is sold as a nutritional
supplement at recommended doses ofup to 4 to 8 g/day to promote the growth of bifidobacteria,
and as an ingredient in nutritional supplement liquids as a source of dietary fiber.
Based on information from FDA's GRAS Notice Inventory 2 website as of April 28, 2015,
the agency has received three notices on FOS and provided "no questions" letters to all of the
notifiers. In May 01, 2000, GTC Nutrition Company submitted GRAS notification (GRN 44) to
FDA for use of FOS in different food categories (GTC Nutrition 2000). On November 22, 2000,
FDA issued "no questions" letter for this GRAS notice (FDA, 2000). Subsequently, two GRAS
notifications were submitted to FDA for use of FOS in infant formulas by: Pfizer Nutrition
(2011; GRN 392) and by Ingredion Incorporated (2014; GRN 537). Both these firms received a
"no questions" letter from FDA (FDA, 2011, 2015). A closely related oligosaccharide, galacto
oligosaccharide, has also been determined to be GRAS for use in a variety of foods in nine
GRAS notifications to the FDA. All these GRAS notices are available at FDA's GRAS Notice
Inventory.
6.1.3.5. scFOS Safety and Degree of Polymerization
6.1.3.5.1. Degree of Polymerization and Fermentability
As described earlier, generally FOS have a degree of polymerization (DP) of 2 to 10 and
can be produced from sucrose by transfructosylation and from inulin by controlled hydrolysis.
Inulin, extracted from chicory roots, has a more heterogeneous DP, ranging from 3 to 60. Given
the large variation in degree of polymerization (DP) of FOS, it is important to address, whether
2 Acee ssib le at: http://www.accessdata.fda.gov/scri pts/fcn/fcnNa vigati on. cfm?rpt=grasListing&disp lay All =true.

the small chain molecules ofscFOS with lower DP can have easier fermentability (and more and
rapid gas formation) or lower fermentability (and lower and slower gas formation).
Rumessen and Gudmand-Hoyer (1998) studied the intestinal transport and fermentation
of chicory-derived long-chain inulin (median DP=12) and oligofructose (median DP=3) in a
single-blind crossover study. In this study, 10 healthy subjects (5/sex; aged 18-25 years) received
single dose tests in random order, separated by 48 hours or more, of 10, 20, and 30 g
oligofructose and 20 g long-chain inulin. Following dose administration, breath samples were
collected every 30 minutes for 12 hours after each test and analyzed for hydrogen (H2). In this
study, hydrogen production profiles were used to estimate orocecal transit times (Table 9). The
investigators concluded that orocecal transit time was slower for the long-chain inulin as
compared to the oligofructose. The fmdings also suggest that the difference is not great as
compared to the extremely large amount of variability. Hydrogen production was not
significantly different between the two fructans, even with substantial differences in DP profiles.
Table 9. Effects of Chicory-Derived Long-chain Inulin and Oligofructose on

HlYd ro2en Pro duction
' andGastromtestina
. . ITrans1t .*
AUC - H2 production Orocecal Transit Time
Test article and dose ppm . min/102 (minutes)
Mean Range Mean Range
Short chain, IO g 139 110-186 105 60-240
Short chain, 20 g 306 241-570 30 15-105
Short chain, 30 g 368 256-615 53 0-165
Long chain, 10 g 247 118-491 75 15- 180
*Adapted from information based on ORN 537
In an in vitro study using human fecal inoculum, Stewart and Slavin (2006) compared the
batch fennentability of 6 chain lengths of inulin and FOS with DP ranging from 2 to >20. In this
study, samples were removed at 0, 4, 8, 12, and 24 hours and total SCFA, acetate, propionate,
and butyrate were measured. The investigators reported, " .. .individual sample chain length did
not follow a clear trend with fennentability," although a statistically significant difference was
detected in the speed of fermentation when the samples were grouped into FOS (DP <10) and
inulin (DP > 10). In another in vitro study, the fermentability of Orafti® GR chicory inulin (DP =
2-60) and Orafti® P95 oligofructose (DP = 2-8) by Lactobacillus rhamnosus, Lactobacillus
acidophi/us, Enterococcus durans, and Enterococcus faecium was compared (Bohacenko et al.,
2013). While the enterococci fermented the shorter chain substrate more efficiently as compared
to the higher-DP oligosaccharide, both species of Lactobacillus fermented both fructans equally
well, leading the authors to conclude that "no significant difference at both lactobacilli species
was observed in respect of utilization ofprebiotics with different chain length."
Based on above described studies, it appears that there is no clear association between
fermentation rate and DP of the oligosaccharide. The available information indicates that as
compared to longer chain oligosaccharides, shorter-chain fructans are fermented rather quickly,
and that there may be differences in the specific small chain fatty acids produced. Based on the
available evidence, this is most likely the case. These differences are so delicate or precise, it is
difficult to analyze or describe, and inconsistent, and highly variable. Additionally, intra
individual variation, and even within an individual over time, is likely a function of many factors
including the luminal pH, the presence of calcium or other buffers, the microbiota profile, etc.
Given this large variability, differences in fermentation of fructans with different DP has only

been shown when the DP difference is extreme, as elucidated by Rumessen and Gudmand
Hoyer's (1998) in comparison of fructans with median DP of 3 and 12 or by Stewart and
Slavin's (2006) in range of DP from 2 to >20-and even in this extreme case, the overall trend
was not statistically significant.
The available information suggest that DP differences among FOS, including scFOS, are
minor. The weighted mean DP of oligofructose (Orafti® P95) and that of scFOS are 4.29 and
3.73, respectively. The median DP of these 2 substances are both= 4. Given all this, it is unlikely
that any difference could be demonstrated in the gastrointestinal handling, fermentation rate,
SCFA production, or fate of these substantially similar substances (polymers). This is not
surprising given that the gut microbiota varies amongst individuals in significant ways.
6.1.3.5.2. Osmolality and Degree of Polymerization
scFOS molecules with low DP are likely to be more osmotically active as compared to
other oligofructoses with higher DP, including the ones used in studies that are used to support or
corroborate the safety of the subject of present GRAS, scFOS. Hence, it is important to address
whether potentially higher osmotic activity from scFOS is not expected to pose a concern,
including increased abdominal distension, pain and laxation, as well as the possibility of severe
laxation/diarrhea. Both breast-fed and bottle-fed infants are currently exposed to human milk and
to infant formula that impose greater osmotic loads than does infant formula with the intended
level of scFOS.
It is well known that human breast milk contains over 200 different oligosaccharides
(Kunz et al. 2000; Vandenplas 2002; German et al., 2008; Ballard and Morrow 2013), with a
total concentration in excess of 1.2 g/100 ml. This level is approximately 3 times higher as
compared to the proposed uses of scFOS in starter formula. The concentration of human milk
oligosaccharides (HMO) has been reported to be 2 g/100 ml of milk on the fourth day of life of
the infant (Vandenplas, 2002). For some mothers, the most dominant component, lacto-N
neotetraose (LNnT; mass 709.3) can be 10 times more intense than the next most abundant lacto
N-fucopentaose I/V (LNFP 1/V; mass 855.3) (Zivkovic et al., 2011). For others, the three most
abundant components, lacto-N-neotetraose, lacto-N-tetraose (LNT; mass 709.3) and lacto-N
fucopentaose IN make up over 50% of the total. Among all samples analyzed to date, a neutral
oligosaccharide with neutral mass 709.3 Da (3Hex, IHexNAc;LNnT) is the most prominent.
Thus the available information suggest that dominant HMOs are short-chain oligosaccharides
with DP 3-5 (the same as scFOS) and with a molecular weight of 709.3 Da, nearly identical with
the 700 Da molecular weight of scFOS.
Chaturvedi et al. (2001) analyzed HMOs from 12 donors and reported that the mean total
oligosaccharide concentration for the 11 typical donors (a total of 77 samples) was
approximately 9 g/L for the first 14 weeks of lactation followed by a gradual decline to
approximately 4 g/L at year one postpartum. These investigators further noted that the
predominant oligosaccharides for the first few months of lactation were 2' -FucLac and LNF-1.
For the first 3 months of lactation, 2'-FucLac, at approximately 3 g/L, was the oligosaccharide
present in the largest concentration. The description of these 2 oligosaccharides was as follows:
2'-FucLac = 2'-fucosyllactose = Fuc-a(l,2)-Gal-P(l,4)-Glc (DP=3); and LNF-1 = lacto-N
fucopentaose-1 = Fuc-a(l,2)-Gal-P(l,3)-Glc-N-Ac-P(l,3)-Gal-P(l,4)Glc (DP=5). All this
information indicates that the predominant HMOs present in human milk at higher levels is
similar to the intended uses of scFOS.

The available information from FDA GRAS notice (GRN 236) and international
regulatory agencies reviews show that galactooligosaccharides (GOS) are commonly used and
have been accepted for addition to infant formula at concentrations as high as 800 mg/100 ml.
Infant formula containing a blend of 90% GOS and 10% long-chain FOS at 800 mg/100 ml has
been in use for many years in the European countries. The GOS added to infant formula actually
has a significantly lower DP as compared to scFOS. As described in GRN 233, a GRAS notice
on combination of GOS and polydextrose (Vivinal®), the DP profile of GOS (mean DP = 3.10)
was as follows: DP2= 33%; DP3= 39%; DP4= 18%; DPS= 7%; DP6, 7, and 8= 3%. The DP
profile (Table 7) of the subject of present GRAS, scFOS by Tata is as follows: DP3= 30%; DP4=
45%; DPS= 5%. Similarly, GOS described in other GRAS notices (GRN 286,489,495; and 569)
for infant formula use have similar DP profiles to Vivinal® GOS. This indicates that GOS added
to infant formula is more osmotically active as compared scFOS. Although the extensive
literature regarding the safety of the addition of GOS to infant formula (at a level twice that
proposed uses for scFOS) was not reviewed in the current notice, it is widely available and has
been provided to FDA in numerous submissions. Thus, the available information indicates that
scFOS supplemented formula is unlikely to cause adverse effects as result of any osmotic
activity.
In summary, human milk-fed infants, as well as infants consuming formula with added
GOS have long been exposed to fluids with higher osmolality as compared to formula containing
the proposed addition level of scFOS with no reported adverse effects. Given this, there is no
reason to expect an adverse osmotic effect with the intended use of scFOS. Additionally, the
osmolality of infant formula is determined by its total formulation, not merely by its
oligosaccharide content (if any). Infant formula manufacturers routinely test formula for
osmolality and adjust its composition as needed to assure that it is within the desired range.
6.1.3.6. scFOS and Changes in Colonic pH
Nilsson and Bjorck (1988) reported significant acid hydrolysis of inulin (fructan) that
increases with time. In addition to this, some animal and human studies also show approximately
10-20% acid hydrolysis of all FOS. It has been also reported that scFOS lowers the colonic pH.
The lowering of colonic pH may lead to more hydrolysis of scFOS in infants. Thus, it is
important to address whether the generation of fructose through increased acid hydrolysis of
scFOS (that could be absorbed) would be of any consequence to infant health, including
increased gastrointestinal discomfort. In the rat study by Nilsson and Bjorck (1988), significant
gastric-acid hydrolysis of inulin has been reported. In a subsequent study, Bjorck and Nilsson
(1991) repeated the study to produce a similar effect (Bjorck and Nilsson 1991). However, this is
a deviant finding that contradicts the far larger body of findings suggesting little or no acid
hydrolysis of fructans during gastric passage. This body of research includes at least two studies
(Bach Knudsen and Hessov 199 5, and Ellegard et al. 1997), in patients with ileostomies. These
studies revealed that little or no digestion or absorption of fructans occurs in either the stomach
or the small intestine. In both of the studies, Bjorck and Nilsson (1991; 1988) it was reported that
a small amount of inulin was apparently hydrolyzed by gastric acid in rats. However, in both
these studies the rats were restricted to only 10 g feed/day, producing abnormally low gastric pH.
It is not clear whether similar phenomenon would be observed, if fructans are consumed ad
libitum or with food - especially with dairy-based food - which is likely to provide significant
buffering. The investigators did not explain in either report why they adopted this approach.

Besides the above reported in vivo experiments in rats, these investigators also performed
an in vitro study in which inulin was incubated for 2 hours in a solution with HCl molarity of
0.10 or 0.05 and observed some degree of acid hydrolysis (Nilsson and Bjorck, 1988). Both the
in vivo and in vitro studies involve a pH of around 1-2, far more acidic than could ever be
produced in the colon by SCF A production, which would be unlikely to reduce the luminal pH to
less than about 5.5. It is also important to note that complete hydrolysis of scFOS produces
substantially less fructose than does hydrolysis of inulin or inulin-derived FOS. This is because
all scFOS includes a glucose endcap, so that, for example, the breakdown of the scFOS DP=3
molecule is only 2/3 fructose and 1/3 glucose. In all, complete hydrolysis of scFOS, with DP=3-
5, produces 72.4% fructose and 27.6% glucose. In contrast, breakdown of inulin with, say,
DP=20, or of FOS derived from such inulin, would be 95% fructose and 5% glucose. Given all
this, free fructose is unlikely to be an issue with scFOS.
6.2. Summary, Discussion and Conclusion
Tata Chemicals Limited (Tata) intends to use small chain fructo-oligosaccharides
(scFOS) as a food ingredient in non-exempt term infant formula. The manufacturing process of
scFOS involves the biotransformation of sucrose by the action of a microbial derived enzyme P
D-fructofuranosidase from Aureobasidium pullulans. The scFOS are prepared using raw
materials and processing aids that are food-grade and comply with applicable U.S. federal
regulations. The scFOS is manufactured according to cGMP in both a liquid (syrup) and powder
form. The scFOS manufactured by Tata is composed of sucrose molecules (glucose-fructose
disaccharides, GF) to which one, two, or three additional fructose units have been added by p 2-1
glycosidic linkages to the fructose unit of sucrose.
Tata has fully developed and characterized the identity and composition of the final
product. scFOS primarily consists of 3 different molecules, each containing a terminal glucose
residue and 2, 3, or 4 fructose residues, designated as GF2, GF3, and GF4. The food grade
specifications for scFOS have been established by Tata. The scFOS in infant formula will be
used at the maximum use levels of 400 mg scFOS/100 ml in starter formula (from birth to
approximately 6 months) as consumed and 500 mg scFOS/100 ml in follow-on formula (infants
older than approximately 6 months) as consumed. The conservative total daily intake of scFOS
by infants is estimated at the 90 th percentile from maximum concentration of 500 mg/100 mL, to
be 1035 mg/kg bw/day. This estimated intake is very conservative for long-term exposure
because as the infant grows the formula intake increases but at a slower rate than weight gain.
Hence, the 90 th percentile intake of scFOS is highest during the first 6 weeks of life and begins to
decline and reach about 840 mg/kg bw/day by 8-12 weeks.
The safety of consumption of scFOS has been supported by several studies conducted on
the metabolism of scFOS and other fructans, as well as safety/toxicity studies in animals and
humans with scFOS and other fructans. Additionally, the safety in use of scFOS is supported
from other GRAS notices on FOS that have been reviewed by FDA and had no questions.
Several fructans, including scFOS, are already GRAS for use in food, including use in infant
formula. In GRN 392, the use of oligofructose was concluded to be GRAS for use in infant
formula, while in GRN 44 (additional uses) and GRN 537 use of scFOS in infant formula was
concluded as GRAS. These uses of scFOS and fructans did not report any adverse effects. In
addition to these GRAS uses in infant formula, several scFOS preparations have GRAS status for
use as a food ingredient in a variety of conventional food and beverage categories (GRN 44, 605,
623,717 and 797) (FDA, 2000; 2016a; 2016b; 2017; 2018). All of these GRAS notifications

have consistently concluded that the addition of scFOS to food is GRAS under their respective
conditions of intended use.
The digestibility of fructans has been investigated in multiple in vitro and animal (rats)
studies. These studies demonstrate that scFOS, unlike like other fructans that are hydrolyzed by
the intestinal enzymes, scFOS is fermented by gut microbiota (Oku et al., 1984; Tsuji et al.,
1986; Tokunaga et al., 1989; Bjork and Nilsson, 1991). Bjork and Nilsson, 1991 reported that
inulin was hydrolyzed in rats by gastric acid due to low gastric pH because of food restriction in
rats to only 10 g feed/day. Therefore, this phenomenon is not expected under conditions of
normal food intake. This is a deviant finding that contradicts the far larger body of research that
has found little or no acid hydrolysis of fructans during gastric passage. Animal studies
demonstrated that scFOS is not absorbed.
In a number of published human studies in healthy adults (Stone-Dorshow and Levitt,
1987; Rumessen et al., 1990; Molis et al., 1996; Alles et al., 1996; Rumessen and Gudmand
Hoyerr, 1998; Castiglia-Delavaud et al., 1998; van Dokkum et al., 1999), as well as in
compromised adults with ileostomy (Bach Knudsen and Hessov, 1995; Ellegard et al., 1997)
effects of scFOS were investigated. The available information from human studies in both
healthy subjects and subjects with ileostomy suggest that majority of ingested fructans, including
scFOS reach the colon where it is fermented by gut microbiota. The kinetics of orocecal transit
time and bacterial fermentation are inversely proportional to the degree of polymerization of the
fructan.
In a summary report on several studies discussed by Carabin and Flamm ( 1999), findings
from the acute toxicity studies suggest that scFOS has a low potential for acute oral toxicity. In a
subacute study, feeding Wistar rats at levels up to 4500 mg scFOS/kg bw/day for 6 weeks did not
produce any adverse effects, as evaluated by hematology, clinical chemistry and histopathology.
Studies in neonatal animals demonstrate the lack of adverse effects of scFOS as discussed by
Carabin and Flamm (1999). Inclusion of scFOS in the diets of neonatal BALB/c mice at 5%
dietary concentration for up to 44 days did not reveal adverse effect on feed intake or body
weight gain (Nakamura et al., 2004). In a study in piglets, Barnes et al. (2012) reported that
addition of 10 g scFOS/L to enteral and parenteral feed of 2-day-old male and female piglets did
not reveal adverse effects on weight gain, weights of stomach, pancreas, liver, and kidney; and
gut morphology. In another study in piglets, Correa-Matos et al. (2003) reported that addition of
7.5 g FOS/L to the colostrum formula fed to 2-day-old piglets revealed enhanced intestinal
function without any adverse effects.
In a repeat-dose 90-day toxicity study by Boyle et al. (2008), Sprague-Dawley rats were
fed standard rodent chow for 13 weeks with 0, 0.55, 1.65, 4.96, or 9.91 % oligofructose, replacing
cornstarch. In this study no treatment related adverse effects as evaluated by food intake, body
weight, body weight gain, clinical observations, hematology, clinical chemistry, or
histopathology even at the highest dose tested, were noted. The NOAEL was the highest dose
tested 4680 mg oligofructose/kg bw/day. In a chronic toxicity and carcinogenicity study (104
week), Clevenger et al. (1988) investigated the effects of feeding of scFOS (at levels up to
50,000 ppm) to male and female Fischer 344 rats. In this study, some statistically significant
differences were noted but there were no toxicologically relevant, test-article-related macro or
microscopic changes in either sex. The incidence of spontaneous tumors in the scFOS-treated
animals was comparable to that of controls, with the exception of pituitary adenomas in male
rats. However, as the pituitary adenoma is one of the most frequently occurring spontaneous

tumors in F-344 rats with highly variable background incidence, the observation was a chance
artifact. The findings from this study suggest that scFOS is not carcinogenic and does not
produce chronic toxicity in rats. Based on the fmdings from this study, the NOAEL was
determined as 50,000 ppm (equivalent to 2170 mg/kg bw/day for males and 2664 mg/kg bw/day
for females, the highest dose tested.
In a reproductive and developmental toxicity study, rats were fed a diet containing 20%
scFOS from day 1 to 21 of gestation. In this study, except for the reduction in body weight of the
pregnant rats and a growth delay for the male pups in the test group during nursing, there were
no other effects on the pregnancy and development of fetuses (Carabin and Flamm 1999). The
reduction in body weight of the pregnant rats appears to be related to lower caloric value for
scFOS, decreased intake of food for this group, or diarrhea observed in the first week and softer
stools in the second and third weeks. The results of this study suggest that feeding of rats at a
20% dietary concentration of scFOS has no significant effects on the course of pregnancy in rats
and on the development of their fetuses and newborns. In another study, also described by
Carabin and Flamm (1999), scFOS at dietary concentrations up to 20% did not adversely affect
the pregnancy outcome or in utero development of the rat. In additional studies, scFOS was
found to be non-mutagenic in bacterial reverse mutation assays, and non-genotoxic in a number
of genotoxicity assay, such as mouse lymphoma assay, unscheduled DNA synthesis (UDS)
assay, and chromosome aberration assay.
In summary, in multiple toxicity studies the safety of scFOS and fructans has been
investigated. These studies included acute, subacute, subchronic, chronic, developmental and
reproductive, and mutagenicity and genotoxicity. Based on the totality of toxicological
information on scFOS and oligofructose it is concluded that the oral toxicity of these substances
is extremely low. In addition to the studies described in animals, the effects of scFOS or
oligofructose (FOS) have been investigated in a number of studies in infants. These studies are
extensively described earlier. Some of the relevant studies in infants are briefly described here.
In a published randomized, double-blind, placebo-controlled trial by Paineau et al.
(2014), 61 healthy term infants aged 0-7 days received formula supplemented with 400 mg/100
ml of either scFOS (DP 3-5) or maltodextrins to the age of 4 months. The scFOS in this study is
substantially equivalent in terms of DP to the scFOS that is the subject of the present GRAS
notice. Formula consumption did not differ between the groups, nor did growth, and the most
frequent adverse event was abdominal pain, followed by liquid stools, but there was no
statistically significant difference in the incidence or severity between the feeding groups. The
findings from this study demonstrates that a milk-based infant formula supplemented with scFOS
at 400 mg/100 ml will increase the fecal content of Bifidobacteria in healthy term infants in
comparison to a placebo formula without inducing any problem of digestive tolerance.
Ripoll et al. (2015) investigated the effect of scFOS on digestive tolerance and growth
parameters in infants up to 10 months of age. In this randomized, controlled, double blind study,
75 formula-fed healthy infants were included at the age of 4 months and received either a
placebo or scFOS (500 mg/100 ml) supplemented formula for six months. The scFOS in this
study is substantially equivalent in terms of DP to the scFOS that is the subject of the present
GRAS notice. Tolerance and growth parameters were similar in both the groups. No difference
was observed between groups for diarrhea and gastroenteritis. The results after 6 months of
supplementation, the strict follow-up of adverse events and digestive tolerance criteria have
demonstrated the good tolerance of scFOS follow-on milk, as no difference was observed

between groups for diarrhea, gastroenteritis, prevalence of infections, regurgitation, constipation
and crying while these conditions are common at this life-stage. The findings from this study,
show that a follow-on milk formula supplemented with 500 mg/100 ml scFOS is safe and well
tolerated leading to normal growth in infants after the age of 4 months and promotes fecal
bifidobacteria levels after one month in never breast fed infants. scFOS addition elicited normal
digestive tolerance and normal growth suggesting it can be used safely at 500 mg/100 ml in
infants after 4 months of age. The findings from this study support the proposed use of scFOS in
follow on formula.
In additional studies in which FOS, but not necessarily scFOS (either high dose or for a
long period of time) further supports the safety of scFOS in infants. In a published randomized,
double-blind, placebo controlled study by Xia et al. (2012), healthy term infants aged :S6 days
were enrolled in a 4-week trial assessing the effects of 4 types of feeding that included cow's
milk (control), human milk (reference), and two FOS groups (240 or 340 mg scFOS/100 ml) on
the intestinal microbiota. The FOS used in this study was confirmed as scFOS. A total of 65
infants completed the study. No differences were reported among groups in stool frequency or
consistency, or in the frequency of feedings with spitups or vomit.
Brunser et al. (2006) compared the effects on infants' fecal microbiota of a standard
milk-based infant formula, the same formula with 200 mg/100 ml of oligofructose, the same
formula with 10 8 cfu L. johnsonii NCC533 (Lal )/g powder, or breast feeding, for a period of 13
weeks, followed by a 2-week washout with standard formula. In this randomized, double-blind
trial of the 116 healthy term infants, 76 formula-fed infants (66% of those enrolled) completed
the entire study. Primary reasons for withdrawal were failure to follow the protocol, antibiotic
use, or illness. The withdrawal rates did not differ across the 3 formula groups and none of the
withdrawals was associated with adverse reaction to the formula. All formulas were well
tolerated.
Bettler and Euler (2006) investigated growth and tolerance of in healthy full-term infants
(aged 14 days or less) fed formula supplemented with oligofructose (150 or 300 mg/100 ml) for
12 weeks in a double-blind study in infants. In this study, overall, at least one adverse event was
reported for 55% of the infants, but the lowest incidence of formula related adverse events was in
the group receiving the higher dose of oligofructose (300 mg/100 ml), and none of the formula
related adverse events was considered to be serious. Additionally, there were no differences
among groups in formula acceptance and tolerance. The investigators concluded that the
experimental cow's milk-based formula supplemented with either 1.5 or 3.0 g oligofructose/L is
safe, well-tolerated and supports normal infant growth. In addition to these studies, several other
published studies in which infants, including preterm infants, were given scFOS or oligofructose
are available and summarized previously. In these studies no adverse effects were reported.
For the present GRAS assessment of scFOS, a comprehensive search of the scientific
literature for safety and toxicity information on scFOS and other similar molecules was
conducted through December 2020 and utilized. Based on the totality of the available published
evidence, it is concluded that there is sufficient qualitative and quantitative scientific evidence to
determine the safety-in-use of scFOS in term infant formula . FOS products have been used in
food for over 18 years with no evidence of adverse effects related to the safety of its use. The use
of a similar manufacturing process in the preparation of the scFOS with similar compositional
analysis, the subject of this GRAS assessment and those that has been the subject of FDA
notifications suggests that the differences between various scFOS products would be limited to

minor variations in the compositional distribution of the FOS oligomers, and to differences in the
residual levels of other sugars. These observations also suggest that the safety information on
FOS products can be interchangeably used. The FDA responses to GRAS notification (GRN
537; GRN 797) on scFOS indicate that the agency is satisfied with the safety-in-use of scFOS at
use levels of 400 mg scFOS/100 ml in starter formula as consumed and 500 mg scFOS/100 ml in
follow-on formula as consumed. The safety determination of scFOS is based on the totality of
available evidence, including current approved uses, in vitro and in vivo metabolism studies, and
a variety of animal studies and, human and infant studies that supports the safety-in-use of
scFOS.
In summary, on the basis of scientific procedures 3, the use of scFOS derived from sucrose
as a food ingredient in infant formula at levels of 400 mg scFOS/100 ml in starter formula as
consumed and 500 mg scFOS/100 ml in follow-on formula as consumed is considered as safe.
The proposed uses are compatible with current regulations, i.e., scFOS is used in infant formula
at use levels of 400 mg scFOS/100 ml in starter formula as consumed and 500 mg scFOS/100 ml
in follow-on formula and is produced according to current good manufacturing practices
(cGMP).
Based on a critical evaluation of the publicly available data described and summarized
herein, Tata Chemicals Limited has concluded that short-chain fructo-oligosaccharides (scFOS),
meeting the specifications cited above, and when used as a food ingredient in infant formula at
use levels of 400 mg scFOS/100 ml in starter formula (from birth to approximately 6 months) as
consumed and 500 mg scFOS/100 ml in follow-on formula (infants older than approximately 6
months) as consumed is Generally Recognized As Safe (GRAS).
It is also the opinion of Tata Chemicals Limited that other qualified and competent
scientists reviewing the same publicly available toxicological and safety information would
reach the same conclusion. Therefore, we have also concluded that short-chain fructo
oligosaccharides (scFOS), when used as described, is GRAS, based on scientific procedures.
3 2 I CFR § I 70.3 Definitions. (h) Scientific procedures include those human, animal, analytical, and other scientific
studies, whether published or unpublished, appropriate to establish the safety of a substance.

7. Part VII- SUPPORTING DATA AND INFORMATION
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APPENDIX I
Analytical results from three lots each for Powder and Liquid batches
Appendix I A: Food Grade Specifications of scFOS Powder (FOS-P95)
Parameters Specification Batch numbers

FOS-P9S FP9D120T04 FP9D120T06 FP9D120T08
Fine white free flowing
Description hygroscopic powder Complies Complies Complies
(clear in solution)
Sweet, without foreign
Taste and Aroma Complies Complies
tastes I odors Complies
Total solids(%) NLT97.0 98.69 98.0 97.90
Moisture (Karl Fisher) (%) NMT 5.0 (w/w) 1.23 2.0 2.10
Residue on ignition
NMT0.l 0.02 0.06 0.02
(sulphated ash) (%)
5.0- 7.S 7.06 5.02 6.33
solution @ 25°C)
(a) Identification
Fructose(% dry basis) NLT67.0 69.57 69.41 71.27
Glucose (% dry basis) NMT33 .0 23.49 22.46 22.73
(b) Assay
Total
NLT95.0 95.25 95.17 95.26
- Trimer (GF2) Informative 35.47 31.05 28.97
--Tetramer (GF3) Informative 50.66 52.21 49.51
-- Pentamer and larger
Informative 9.14 11 .96 16.99
(GF4 and higher)
NMT5.0 4.75 4.83 4.74
+ Fructose
Heavy metals
Lead (as Pb) (ppm) NMT0.02 <0.02 <0.02 <0.02
Arsenic (as As2O3) (ppm) NMT0.1 <0.05 <0.05 <0.05
Cadmium (Cd) (ppm) NMT0.01 <0.01 <0.01 <0.01
Mercury (as Hg) (ppm) NMT0.01 <0.01 <0.01 <0.01
Chromium (as Cr) (ppm) NMT0.0S <0.05 <0.05 <0.05
Tin (as Sn) (ppm) NMT50 <0.50 <0.50 <0.50
Copper (as Cu) (ppm) NMT30 <0.50 <0.50 <0.50
Methyl Mercury
(Calculated as the element) NMT0.25 <0.02 <0.02 <0.02
(ppm)
Microbiololdcal limits
Total Plate Count (cfu/g) NMT300 < 10 < 10 < 10
Enterobacteriacea (MPN/g) NMT3.0 <3 <3 <3
Yeasts & Mould (cfu/g) NMT20 < 10 < 10 < 10
Escherichia coli (MPN/g) Absent IO g Absent Absent Absent
Tata Chemicals Page 60of63 FOS- IF-GRAS

FOS-P95 FP9D120T04 FP9D120T06 FP9D120T08
Staphylococcus aureus Absent 10 g Absent Absent Absent
Salmonella spp Absent 100 g Absent Absent Absent
Shigella spp Absent 25 g Absent Absent Absent
Listeria monocytogenes Absent 25 g Absent Absent Absent
Sulphite reducing
NMTlO < 10 < 10 < IO
Clostridia (cfu/g)
Cronobacter sakazakii Absent300 g Absent Absent Absent
Bacillus cereus (cfu/g) NMT 100 < 10 < 10 < 10
Mycotoxins
Ajlatoxin Bl (ppb) NMT0.5 <0.5 <0.5 <0.5
Ajlatoxin Bl (ppb) NMT0.5 <0.5 <0.5 <0.5
Ajlatoxin Gl (ppb) NMT0.5 <0.5 <0.5 <0.5
Ajlatoxin G2 (ppb) NMT0.5 <0.5 <0.5 <0.5
Ajlatoxin Ml (ppb) NMT0.025 <0.022 <0.024 <0.023
Melamine (ppm) NMT0.5 <0.5 <0.5 <0.5

Appendix I B: Food Grade Specifications of scFOS Liquid (FOS-L95)

FOS-L95 FL9T220003 FL9T220005 FL9T220007
Colorless to sunshine Light Yellow Colourless
Colourless
Description yellow color syrupy color syrupy syrupy liquid
syrupy liquid
liquid liquid
Sweet, without foreign
Taste and Aroma Complies Complies Complies
tastes / odors
Moisture (Karl Fisher)(%) NMT25 22.90 24.22 22.38
Brix (Refractometer) 0 Bx NLT75 75.85 76.91 75.67
Residue on ignition
NMT0.l 0.05 0.06 0.04
(sulphated ash)(%}
5.0 - 7.5 6.26 5.93 5.88
solution @ 25°C)
(a) Identification
Fructose(% dry basis) NLT67.0 68.30 68.67 67.38
Glucose (% dry basis) NMT33.0 26.73 26.42 26.91
(b) Assay
Total
NLT 95.0 95.30 95.47 95.11
-- Trimer (GF2) Informative 50.34 46.73 51.3
-- Tetramer (GF3) Informative 40.60 43.38 39.87
-- Pentamer and larger
Informative 4.36 5.37 3.94
(GF4 and higher)
NMT 5.0 4.70 4.53 4.89
+Fructose
Heavy metals
Lead (as Pb) (ppm) NMT0.02 <0.02 <0.02 <0.02
Arsenic (as As) (ppm) NMT0.1 <0.03 <0.03 <0.03
Cadmium (Cd) (ppm) NMT0.01 <0.01 <0.01 <0.01
Mercury (as Hg) (ppm) NMT0.01 <0.01 <0.01 <0.01
Chromium (as Cr) (ppm} NMT0.05 <0.05 <0.05 <0.05
Tin (as Sn) (ppm) NMT 50.0 <0.05 <0.05 <0.05
Copper (as Cu) (ppm) NMT30.0 <0.13 <0.09 <0.05
Methyl Mercury
(Calculated as the element) NMT0.25 <0.01 <0.01 <0.01
(ppm)
Microbiological limits
Total Plate Count (cfu/g) NMT 300 < 10 < 10 < 10
Enterobacteriacea (MPN/g) NMT3 <3 <3 <3
Yeasts & Mould (cfu/g) NMT20 < 10 < 10 < 10
Escherichia coli (MPN/g) Absent 10 g Absent Absent Absent
Staphylococcus aureus Absent 10 g Absent Absent Absent
Salmonella spp Absent 100 g Absent Absent Absent
Shigella spp Absent25 g Absent Absent Absent

FOS-L9S FL9T220003 FL9T22000S FL9T220007
Listeria monocytogenes Absent25 g Absent Absent Absent
Sulphite reducing < 10 < 10
NMTlO <10
Clostridia (cfu/g)
Cronobacter sakazakii Absent 300 g Absent Absent Absent
Bacillus cereus (cfu/g) NMT 100 <10 < 10 < 10
Mycotoxins
Aflatoxin Bl (ppb) NMT0.5 <0.5 <0.5 <0.5
Aflatoxin Bl (pph) NMT0.5 <0.5 <0.5 <0.5
Aflatoxin G1 (ppb) NMT0.5 <0.5 <0.5 <0.5
Aflatoxin G2 (ppb) NMT0.5 <0.5 <0.5 <0.5
Aflatoxin Ml(ppb) NMT0.025 <0.023 <0.021 <0.023
Melamine (ppm) NMT0.5 <0.5 <0.5 <0.5
Tata Chemicals Page 63 of63 FOS-IF-GRAS

From: Madhu Soni
To: Morissette, Rachel
Cc: "Dipak Bagad"
Subject: RE: [EXTERNAL] RE: questions for GRN 990
Date: Monday, July 19, 2021 10:52:51 AM
Attachments: image001.png
scFOS GRAS infant formula-GRN 990-FDA Query responses final.pdf
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. Morissette,

As mentioned, please find attached a pdf copy of the responses to FDA questions raised by
your team for GRN 990. We apologize for the slight delay. If you need any further
clarification please let me know.
Thank you for this opportunity.
Best regards,
Madhu
------------------------------------------
Madhu Soni, PhD, FACN, FATS
Soni & Associates Inc
749 46th Square
Phone: +1-772-299-0746
Cell: +1-772-538-0104
www.soniassociatesnet
From: Morissette, Rachel [mailto:Rachel.Morissette@fda.hhs.gov]
Sent: Monday, July 19, 2021 8:23 AM
To: Madhu Soni <sonim@bellsouth.net>
Subject: RE: [EXTERNAL] RE: questions for GRN 990
Dear Dr. Soni,
We have not received responses to our questions for GRN 000990 yet. Can you please let me know
when you except to send those?
Thank you for your consideration.
Rachel
-------------------------------------------------------------
Rachel Morissette, Ph.D.
Regulatory Review Scientist
Division of Food Ingredients

rachel.morissette@fda.hhs.gov
1
11 U.S. FOOD & DRUG
ADM IN ISUAT I ON
D C1 m ··
From: Madhu Soni <sonim@bellsouth.net>
Sent: Saturday, July 3, 2021 8:06 AM
To: Morissette, Rachel <Rachel.Morissette@fda.hhs.gov>
Subject: [EXTERNAL] RE: questions for GRN 990
Thank you Dr. Morissette for the email and the FDA queries
I will try my best to get you the responses in time.
Best regards. Have a great 4th of July weekend.
Madhu
Sent: Friday, July 2, 2021 12:38 PM
Subject: questions for GRN 990
Dear Dr. Soni,
Please see attached our questions for GRN 990. Have a good weekend.
Best regards,
Rachel
-------------------------------------------------------------

1
11 U.S. FOOD & DRUG
ADM IN ISUAT I ON
D C1 m ··
RE: GRN 990 (short-chain Fructo-oligosaccharide GRAS notice)
This responds to your email of July 2, 2021 regarding clarifications required for our short-
chain fructooligosaccharides (scFOS) GRAS notice (GRN 000990) for use as an ingredient in
cow milk-based, non-exempt infant formulas for term infants, as consumed, at a level up to 400
mg scFOS/100 mL starter formula and up to 500 mg scFOS/100 mL follow-on formula. We are
providing a point-by-point response to your queries along with some relevant
clarifications/discussion.
Regulatory:
FDA Query 1. The notice refers to many prior GRNs, along with the dates
and notifiers for those GRNs. There are a number of instances throughout
the notice where the GRNs do not line up with the date of the response
letter or notifier cited for that notice in the GRAS notice inventory, which
makes it difficult to determine which notice is actually being referenced.
Additionally, in at least one example, a notice is cited for a completely
unrelated ingredient (i.e., p. 39 cites GRN 979). Please go through the
notice and correct any errors when citing prior GRNs.
Response: We are sorry for the oversight. The GRN 979 should have been GRN 797. We
have checked all the GRN citations and a list with correct links is given in the attached Table
1 (please see Appendix I). The confusion appears to be due to changes to the FDA GRAS
Notice inventory website and we did not check the links that were used earlier in our
previous Tata Chemicals GRAS notification on FOS (GRN 605) for uses in conventional
foods. Again please accept our apology for the oversight.
FDA Query 2. We note that the notice discusses health benefits related to
the use of Tata Chemicals’ scFOS. In those discussions, the terms
“prebiotic” and “probiotic” are used several times throughout the notice.
As you are aware, we do not evaluate any purported health benefits in a
GRAS notice, and FDA does not have regulatory definitions for “prebiotic”
and “probiotic”. Please remove any references to those terms in the
notice.
Response: We agree with FDA suggestion that in the absence of a regulatory definition and
given the purported health benefits, we request to remove the terms “prebiotic” and
Page 1 of 22
“probiotic” from the GRAS notification. We note that the prebiotic term appeared 20 times
(including 6 times in references) in the GRAS notice and we agree to remove the term
“prebiotic” from the description except those from the reference list. The “probiotic” term
appeared three times in the GRAS notice, including once in the reference list. We agree to
remove the term “probiotic” except the one mentioned in reference list.
FDA Query 3. On p. 13 of the notice, the citation for sodium hydroxide is

listed as 21 CFR 184.1631; however, this regulation is for the use of
potassium hydroxide. Please provide the correct citation for sodium
hydroxide.
Response: Sorry for the oversight, the correct citation for sodium hydroxide is as follows:
21 CFR 184.1763
FDA Query 4. The terms “starter formula” and “follow-on formula” are used
in the notice. FDA does not have a definition for these terms. Please
specify the intended age ranges in months for these intended uses.
Response: The intended age ranges for the proposed use of scFOS are as follows: at a level
up to 400 mg scFOS/100 mL formula for infants 0-6 months and up to 500 mg FOS/100 mL
formula for infants >6 months of age.
FDA Query 5. On p. 14 of the notice, the citation “Femon (1993)” is used.

However, there is no author with this name in the reference section.
Please clarify if “Fomon (1993)” is intended instead.
Response: Thank you for bringing this to our attention and we are sorry for the oversight.
We intended to use Fomon (1993).
FDA Query 6. On p. 10 of the notice, Tata Chemicals states that the β-

fructofuranosidase enzyme used in the manufacture of scFOS is
membrane bound. However, this aspect of the process is not elaborated in
the manufacturing section of the notice. Instead, Tata Chemicals states
that the process involves the use of the culture biomass that contains the
β-fructofuranosidase enzyme. Please clarify the use of the enzyme and
Page 2 of 22
whether it is membrane bound and if it is expected to be in the final
product.
Response: Please note that the enzyme that catalyzes the reaction in the production process
of scFOS manufacturing is an intracellular enzyme, which is present in the cell membrane
and doesn't get released in the reaction mixture. Hence, Tata Chemicals used culture biomass
instead of the free enzyme for the conversion of cane sugar to scFOS. Once the reaction is
completed the culture biomass is removed by fine filtration. In this process, the enzymes are
not present in the final product.
Fine Filtration: A four stage filtration system is used for the separation of the biomass. In the
first stage, culture biomass is filtered and separated by a plate filtration system with a 20
micron pore size cloth as the filtering membrane. In the second, third and fourth phases,
respectively, 15 micron, 5 micron and 3 micron filter membranes are used to remove any
residual fine parts of the biomass or any other particles, precipitate, etc.
FDA Query 7. Please confirm that the enzyme is GRAS for its intended use
and meets the Joint FAO/WHO Expert Committee on Food Additives and
Food Chemicals Codex (FCC) specifications for enzymes used in food.
Response: As mentioned above, please note it is not the enzyme but the biomass is used.
Tata chemicals consider that the biomass containing the enzyme, used in the production of
scFOS is GRAS. Please note that this is the same biomass containing the enzyme that was
used in Tata Chemicals GRAS notification on FOS (GRN 605) for its use in conventional
foods in which FDA did not object to the use of the biomass.
The biomass used in the scFOS production as a source of enzyme is from the microorganism
Aureobasidium pullulans, the same microorganism used in the earlier GRAS notification
GRN605. This microorganism is well known for its application in biotechnology, such as
production of Pullulan and beta-glucan (GRN 99, 605 and 309). It is yeast like fungi and
generally recognized as safe. Our toxicology study of the scFOS further conferred its safety
in production of food ingredients (Jain et al., 2019).
FDA Query 8. In describing the methodologies used for specifications,

please provide complete citations for referenced methods and provide
confirmation that all methods, including “in house” methods, are validated
and appropriate for the respective analytes.
Page 3 of 22
Response: We confirm that all methods, including “in house” methods, are validated and
appropriate for the respective analytes. FOS analysis is done as per FCC 11th edition (year
2018) page no. 499 to 500.
Please note that, we have got the testing’s done through accredited laboratory (SGS India
Private Limited) and below are the details of the test parameters and respective validation
status.
Validation
Test Parameter SGS Internal SOP Base Reference method
status
Lead
Arsenic SO-IN-MUL-TE-063A
AOAC 2015.01 and AOAC 2015.06 Yes
Cadmium By ICPMS
Mercury
1. Metals and other elements in plants and pet
foods – AOAC 985.01
Chromium 2. Metals in plants and pet foods – AOAC 975.03
3. Metals in solid waste – AOAC 990.08
4. Method for fortified foods-AOAC 2011.14
SO-IN-AFL-MNR-C-
5. Method for heavy metals in food-AOAC Yes
Tin TE-006
2013.06
6. Heavy Metals In food AOAC Official method
Copper
2015.01
7. Method for Minerals & Trace Elements- AOAC
Methyl Mercury
2015.06
SO-IN-AFL-MNR-C- Instruction Manual of RIDASCREEN Aflatoxin
Aflatoxin M1 Yes
TE-065 M1 Art No.-R1121
Simultaneous determination and confirmation of
melamine and cyanuric acid in animal feed by
zwitterionic hydrophilic interaction
chromatography and tandem mass spectrometry.
David N. Heller and Cristina B. Nochetto, Rapid
Commun. Mass Spectrom., Volume 22, Issue 22,30
November 2008 ,Pages 3624–3632
LIB No. 4421,Volume 24, October 2008, Division
of Field Science, Office of Regulatory Affairs, U.S.
Food and Drug Administration Determination of
SO-IN-AFL-MNR-C- Melamine and Cyanuric Acid Residues in Infant
Melamine Yes
TE-023 Formula using LC-MS/MS
ISO/TS 15495:2010 (IDF RM 230:2010) Milk,
Milk Products And Infant Formulae -- Guidelines
For The Quantitative Determination Of Melamine
And Cyanuric Acid By LC-MS/MS
Laboratory Information Bulletin LIB No.
4422,October 2008, Division of Field Science,
Office of Regulatory Affairs, U.S. Food and Drug
Administration, Interim Method for Determination
of Melamine and Cyanuric Acid Residues In Foods
using LC-MS/MS
Page 4 of 22
FDA Query 9. Please clarify when specifications are on a dry basis. For
example, limits for fructose and glucose levels are listed on a dry basis;
however, other specified limits, such as total fructooligosaccharides and
other carbohydrates, do not include that designation.
Response: Please note that the Specifications provided in the Tata Chemicals notice are all
on the dry basis.
FDA Query 10. We note that the specifications for 1-kestose, nystose, and
fructofuranosylnystose are listed as “informative.” Please elaborate on this
parameter and provide quantitative limits if applicable. We note that FCC
specifications for scFOS include limits for trimer (≥ 30.0%), tetramer (≥
45.0%), and pentamer and larger (≥ 5.0%).
Response: All the batches follow the FCC 11th edition (year 2018) page no. 499 to 500
specifications. While in the shared batch no. FP9D120T08, the trimer (GF2) value is 28.97%
(Below 30%), we will ensure that all future batches fall within the FCC specifications. Please
find below the data of additional batches that meet the FCC Specifications.
Purity Specific FP9D1 FP9D1 FP9D1 FP9D1 FP9D1 FP9D1 FP9D1 FP9D1 FP9D1 FP9D1 FP9D1
(%) ation 21035 21036 21037 21038 21039 21040 21041 21042 21043 21044 21045
GF2 NLT 30 38.63 38.33 38.39 39.63 41.24 39.63 38.28 37.61 38.7 37.95 38.14
GF3 NLT 45 48.91 49.26 49.03 48.42 47.58 48.42 48.95 48.93 48.14 48.43 48.36
GF4 NLT 5 7.93 8.18 8.25 7.82 7.38 7.82 8.34 8.62 8.49 8.89 8.72
Purity NLT 95 95.47 95.77 95.67 95.87 96.2 95.87 95.57 95.16 95.33 95.27 95.22
Please note that for the liquid samples, while maintaining GF2, GF3 and GF4 ratio, it was
leading to crystallization; hence, these specifications have been referred as informative.
FDA Query 11. The notice includes specified limits for methyl mercury,
melamine, and aflatoxins, as well as relatively high limits for tin and
copper compared to the results of the batch analyses. Please discuss
whether Tata Chemicals has reason to expect the presence of these
substances in scFOS.
Page 5 of 22
Response: Please note that in the Tata Chemical GRAS notice, specified limits for heavy
metals and Aflatoxin are on the basis of Food Safety and Standard Authority of India
(FSSAI, 2020) Food Safety Standards (Contaminants, Toxins and Residues) Regulations
2011, where limits for Fructo-oligosaccharides are not mentioned; hence, the limits for
“Foods not specified” or “All foods” is taken into consideration for establishing the
specification parameters. In addition to this, we have also considered other international
markets for establishing these specifications. We do not have any particular reason to expect
the presence of these substances in scFOS. Nevertheless, Tara Chemicals intends to test for
these substances on a periodic basis to ensure their absence or very low levels in our
products.
FDA Query 12. Please confirm that the batch analyses of powder and liquid
scFOS that are reported in Appendix I of the notice are non-consecutive
lots of scFOS.
Response: We confirm that the batch analysis of powder and liquid reported in Appendix I
and II are from non-consecutive lots of scFOS.
FDA Query 13. On p. 13 of the notice, Tata Chemicals states that “The
resins and microfiltration used are in compliance with FDA guidelines.”
Please provide a citation for these guidelines and confirm that all materials
and processing aids meet applicable U.S. regulations for use in the
production of food ingredients.
Response: The resin and microfiltration used as per the guidelines provided in the CFR-21
document -Ref is as below:
Resins:
Resin complying with FDA in Code of Federal Regulations 21 CFR 173.25.
Microfiltration:
The materials of construction meet the FDA requirements for food contact use as detailed in
Code of Federal Regulations, 21 CFR paragraphs 170-199 in that:
• Polypropylene to 21 CFR section 177.1520 (Olefin polymers)
• Ethylene Propylene Rubber and Silicone Elastomeric seal materials to 21 CFR section
177.2600 (Rubber articles intended for repeated use, excluding milk and edible oils)
FDA Query 14. In the notice, Tata Chemicals discusses potential dietary
exposure to scFOS from intended uses other than infant formula. The
Page 6 of 22
notice states that as infants grow and begin to consume other foods,
infant formula intake decreases, along with exposure to scFOS due to
other foods being unlikely to contain scFOS at levels comparable to infant
formula. However, this point regarding the use level in other foods is
incomplete, since it does not address the amount of scFOS-containing
food consumed in the infant background diet. Please address the
cumulative dietary exposure to scFOS in infants resulting from both the
intended use in infant formula and the background uses of scFOS in
conventional foods that have been described in other GRAS notices. For
example, GRN 000717 includes the use of scFOS in infant and toddler
foods in addition to infant formula, as well as other foods that may be
consumed.
Response: We note that to some extent this query related to intake of scFOS from
background and proposed uses has been addressed in an earlier GRAS notice. Following
completion of GRN 44 in 2000 and subsequently in 2007, GTC notified FDA (additional
correspondence) that it had determined that the addition of scFOS to foods in general,
including infant and toddler foods but excluding infant formula, at levels resulting in intakes
up to 20 g/day in the general population and up to 4.2 g/day in infants less than one year of
age, is also GRAS. Following its review, FDA (2007) had no questions regarding this
conclusion. Also, please note that the proposed use levels of scFOS in infant formula (starter
or follow-on) by Tata Chemicals is at the same levels as described by Ingredion (2014) in
GRAS notice (GRN 537) and NFBC in GRAS notice (GRN 797). Also, the subjects of these
three GRAS notices (including GRN 990) contains the same levels of scFOS, i.e., 95%.
Given this, the intake of scFOS for formula (only) fed infants will be same and there will not
be any increase in the overall consumption of scFOS resulting from this use. As regards
infants who start consuming complimentary foods, the intake of scFOS from infant formula
will be reduced. Thus there will be a decrease in the level of scFOS consumed. The amount
of scFOS-containing food consumed in the infant background diet is likely to differ.
However, it is unlikely to be of any safety concern. As indicated earlier scFOS has been
determined to be safe at levels up to 4.2 g/day in infants less than one year of age.
As scFOS manufactured by Tata Chemicals will serve as an alternative source of scFOS to
existing GRAS sources of scFOS described in GRN 44, GRN 537, GRN 797 and GRN 717
(including infants less than 1 year old), the introduction of scFOS by Tata Chemicals is
unlikely to further increase dietary intake of scFOS in an additive manner. The proposed uses
of scFOS by Tata Chemicals will serve as an alternative to existing GRAS sources and,
therefore, will not change the current dietary exposure to scFOS among U.S. consumers of
foods to which FOS may be added. Any additional intake is considered as safe.
Toxicology
Page 7 of 22
FDA Query 15. On p. 18 of the notice, Tata Chemicals states, “In this GRAS
assessment, attempts have been made to summarize the available
information, related to safety of FOS, in the order of their importance”
(emphasis added). However, in Section 6.1.1 (Pivotal or Primary Published
Clinical Studies of scFOS in Infant), the initial study described is Guesry et
al., 2000, which the notice indicates has been published as an abstract.
The second and third studies described, Lasekan et al., 2015 and Xia et
al., 2012, with tested use levels for scFOS below the levels proposed by
Tata Chemicals.
We note that data published as an abstract cannot be considered primary
evidence for a GRAS conclusion. Similarly, it is unclear how Lasekan et al.,
2015 and Xia et al., 2012 can be considered pivotal studies to support the
intended use given the use levels are below Tata Chemicals’ proposed use
level.
Additionally, on p. 50 of the notice in Section 6.2 (Summary, Discussion and
Conclusion), Tata Chemicals states, “Some of the relevant studies in
infants are briefly described here.” However, the initial studies discussed
in this section are Paineau et al., 2104 and Ripoll et al., 2015. The studies
by Guesry et al., 2000 and Lasekan et al., 2015 are not summarized in
this section of the notice.
Therefore, it is unclear which infant studies Tata Chemicals considers to be
pivotal to the GRAS conclusion. Please provide a discussion that addresses
which infant studies are considered pivotal to the current GRAS conclusion
and why.
Response: Sorry for our confusion regarding the use of the term “pivotal” in the GRAS
dossier and describing the studies accordingly. Instead of pivotal we should have just
described the available relevant studies and its support to the GRAS assessment based on all
available evidence. As mentioned in the GRAS notice, the safety determination of scFOS for
use in infant formula at the proposed use levels is based on the totality of the available
evidence, including current approved uses, in vitro and in vivo metabolism studies, and a
variety of animal studies and human and infant studies that supports the safety-in-use of
scFOS.
We agree that Guesry et al. (2000) study is published as an abstract and can only be
considered as supportive evidence. Similarly, the studies by Lasekan et al. (2015) and Xia et
al. (2012) cannot be considered as pivotal as the levels of scFOS tested in these studies are
below the levels proposed by Tata Chemicals. However, given this, these studies also provide
the supportive evidence and are not pivotal.
In Section 6.2., we missed mentioning about two relevant studies by Guesry et al. (2000) and
Lasekan et al. (2015). Although published as an abstract, the study by Guesry et al. (2000)
supports the safety of the proposed uses of scFOS. The study by Lasekan et al. (2015) used
lower doses as compared to the proposed doses; however, no adverse effects were noted. The
Page 8 of 22
findings from this study indicate that scFOS is unlikely to cause adverse effects. We would
like to incorporate both these studies described in Section 6.1.1. as supportive evidence in the
Summary Discussion and Conclusion section 6.2.
We apologize for creating this confusion. We request that the agency consider the totality of
the available evidence as the basis to support the present GRAS assessment as we believe
that all of the cited studies contribute to the overall safety of scFOS at the intended use
levels.
FDA Query 16. On p. 18 of the notice, Tata Chemicals indicates that non-
digestible oligosaccharides, including FOS, may decrease serum lipids,
including cholesterol. On pp. 32 and 36 of the notice, Tata Chemicals
indicates that there were decreases in cholesterol reported in rats
following consumption of scFOS (i.e., Jain et al., 2019 and Takeda and
Niizato, 1982). Similarly, on p. 42 of the notice, when discussing
corroborative studies in infants, Tata Chemicals states, “Levels of total
cholesterol in blood were significantly higher in the human milk group
than in either formula group….” Please provide a brief narrative that
specifically discusses the impact, if any, of scFOS consumption on serum
cholesterol levels in infants and why this is not expected to be a safety
concern.
Response: The available evidence from rat studies indicate that exposure to scFOS results in
decreased levels of cholesterol (Jain et al., 2019; Takeda and Niizato, 1982). However, the
findings from unpublished study in infants fed human milk revealed increase in blood
cholesterol levels, while in infants fed formula (with and without scFOS) no such increase
was noted. The publicly available information shows that breast milk contains more
cholesterol as compared to infant formula (Friedman and Goldberg, 1975) and breastfed
infants have higher blood cholesterol (Owen et al., 2002; Wong et al., 1993). It has been also
reported that higher neonatal dietary cholesterol is associated with different cholesterol
metabolism and less endogenous cholesterol synthesis in infants who are breastfed (Wong et
al., 1993; Demmers et al., 2005). There is lack of evidence as to whether a change in
synthesis or metabolism of cholesterol in the neonatal period persists beyond weaning and
into adulthood (Demmers et al., 2005). The available evidence indicates that scFOS is
unlikely to be of safety concern. Thus, we would consider any impact to be negligible.
Please note that the study discussed on page 42 was an unpublished study conducted by
Abbott (1993) and reported in the previous GRAS notice GRN 537 (Ingredion, 2014).
Additional details of this study were not available for independent review.
Page 9 of 22
FDA Query 17. (a) On p. 26 of the notice, Tata Chemicals states “The first
GRAS notice on scFOS, GTC Nutrition (2000) established the ADI of 4.2
g/day scFOS for infant [sic] (<1 year old).” However, we note that the
dietary exposure to scFOS from the intended use in infant formula at
the 90th percentile may exceed the stated ADI depending on body
weight (bw). Please provide a safety narrative that compares total
dietary exposure to scFOS to Tata Chemicals’ stated ADI and discuss
why it does not pose a safety concern for infants. Please provide a
safety narrative that discusses why consuming scFOS at levels above
Tata Chemicals’ stated ADI does not pose a safety concern for infants
aged 0-6 months.
Response: It should be noted that the ADI of 4.2 g/day for scFOS in infant reported in GRN
44 was determined based on a 1987 survey of over 20,000 infants. In this survey, the safety
and tolerance of FOS consumption in infants was examined (Yamamoto and Yonekubo,
1993; cited in GRN 44). Based on the FOS concentrations reported in Japanese infant
formula and estimates of formula intake in the U.S., the mean and 90th percentile FOS
intakes were estimated to be 3.0 and 4.2 g FOS/day, respectively. In this survey, no
statistically significant differences between breastfed infants and those fed formula were
observed for growth, mothers’ perception of health of the baby, or any other adverse effects
included in the survey. This shows that the ADI was established based on findings from EDI.
The actual ADI has not been determined and may be higher. It is almost 34 years, since the
recognition of the ADI of 4.2 g/day and the infant formulas containing FOS are still marketed
without any safety concerns. Thus, the accumulating evidence for over three decades indicate
that the proposed use of scFOS in infant formula and its addition to baby foods for infants is
unlikely to result in adverse effects.
As described in our GRAS notice (GRN 990), the 90th percentile EDI from the proposed uses
of scFOS in “starter” and “follow-on” infant formula ranges from 828 to 1035 mg/kg
bw/day, respectively. As described in GRN 44, for infants 5 through 11 months the 90th
percentile intake is estimated as 3.1 g/day (337 mg/kg bw/day). This additional intake of
scFOS is unlikely to be additive, as the infants starts the intake of complimentary foods that
may contain scFOS, the intake of scFOS from infant formula decreases. It is also unlikely
that the total intake of scFOS from complimentary foods will significantly increase and will
be of safety concern in infants receiving infant formula and complimentary foods. The EDIs
determined in the GTC Nutrition GRAS notice (GRN 44) assume that scFOS will be used at
the proposed use level in all 18 food categories to which scFOS is intended to be added. As
such, the EDIs derived are considered highly conservative estimates of potential scFOS
intake. Thus, any additional intake of scFOS from complimentary formula is considered as
safe. The proposed use levels by Tata Chemicals in infant formula are identical to those
described in GRN 797 and GRN 537, and both these GRAS notices received a “no
questions” letter.
Page 10 of 22
Query 17. (b) In Table 7 (p. 40 of the notice), Tata Chemicals lists the
ADI for scFOS as “At proposed use levels (4 or 5 g/L)” for the current
notice, as well as for GRNs 000797 and 000537. This statement is
unclear, as ADI values are usually expressed in mg/kg bw/d. Please
provide an explanation that clarifies this statement.
Response: Sorry for the confusion, as such in both of these GRAS notices (GRN 797 and
GRN 537), the ADI values were not established. The safety was established for the
intended use level of 400 mg/100 ml (4 g/L) for “starter formula” (within the first month of
life) that results in a 90th percentile intake of 828 mg/kg bw/day and at 500 mg/100 ml (5
g/L) in “follow-on formula” (infants older than 1 month) that results in the 90th percentile
intake of scFOS is about 800 mg/kg bw/day. In both of these GRAS notices, based on the
totality of the evidence, the notifiers concluded that the intended use of scFOS in term
infant formulas is GRAS.
FDA Query 18. In several sections of the notice (listed below), Tata
Chemicals incorporates into the notice data and information from previous
notices. However, we note that each GRAS notice must independently
support the safety of the notified ingredient for its intended use. For each
study Tata Chemicals considers critical to the GRAS conclusion for scFOS
in infant formula for term infants and that they intend to incorporate into
the notice, please provide a summary of the study, along with the
complete citation and the specific GRAS notice from where the study
came.
Page 26: “These studies have been the subject of several comprehensive
evaluations, including several GRAS notices [GRN 44 (FDA, 2000), 537
(FDA, 2015), 605 (FDA, 2016a), 623 (FDA, 2016b), 717 (FDA, 2017), 797
(2018)] that have been reviewed by independent expert panels and the
FDA. Among these GRAS notices on scFOS, GRN 605 was submitted by
Tata. As the available information is extensively described in these
previous GRAS notices, including GRN 605 Tata, all these GRAS notices
are incorporated in the present GRAS by reference.”
Page 39: “Tata is hereby incorporating all the toxicology and human
tolerance studies discussed in these previous GRAS notices by reference
(NFBC, 2018; Galam, 2017; NFBC, 2016; Tata, 2015; Ingredion, 2014;
Pfizer, 2011; GTC, 2000).”
Page 40: “Given the structural and chemical similarity of scFOS preparations
that have been concluded GRAS (e.g., GRN 797 and 537) by NFBC (2018)
and Ingredion (2014) with the current GRAS (Table 7), a discussion of
Page 11 of 22
publicly available data and information relevant to the safety of scFOS is
incorporated by reference to studies described in GRN 797 and 537.”
Response: We are sorry for the lack of our understanding as regards citing the previous
GRAS notices that were submitted to FDA by other notifiers and received no question letters.
We agree that each GRAS notice must independently support the safety of the notified
ingredient for its intended use. Please note that all relevant data and safety studies mentioned
in all these GRAS notices and critical to the GRAS conclusion for scFOS in infant formula
for term infants are appropriately described in our GRAS notice (GRN 990). As per our
understanding there are no additional studies or information in these previous GRAS notices
that is not described in our GRAS notice.
FDA Query 19. On p. 18 of the notice, the intake of scFOS in the Guesry et
al., 2000 study is given as 1, 2, or 3 g/day. However, in Table 2 (p. 19 of
the notice), the intakes for this study are listed as 200, 400, or 600
mg/day. Please clarify this discrepancy.
Response: Thank you for bringing this to our attention and sorry for the oversight. In this
study, Guesry et al. (2000) compared the effects of 3 concentrations of scFOS in infant
formula. Infants received 5 bottles of formula per day for 2 weeks; each bottle provided
either 200 mg lactose or 200, 400, or 600 mg scFOS providing daily intakes of 1.0 g lactose
or 1.0, 2.0, or 3.0 g scFOS/day. In Table 2 the intake values should have been 1000, 2000 or
3000 mg/day. Please accept our apology for the discrepancy.
FDA Query 20. On p. 26 of the notice, Tata Chemicals states that updated
literature searches were conducted to identify new studies relevant to the
safety of scFOS in children and adults. However, no end date was
provided for the search. Please provide an end date (i.e., month and year)
through which Tata Chemicals searched the published literature.
Response: The end date for the updated searches was October 2020. Sorry, we forgot to
mention this.
FDA Query 21. In Section 6.1.2.3. (scFOS Studies in Piglets and Other
Weaning Animals), Tata Chemicals discusses studies in piglets and other
weaning animals. For studies in which the test article was administered in
Page 12 of 22
the feed (or drinking water) to piglets, rats, or mice (see below), please
provide equivalent dose levels on a bw basis (i.e., mg/kg bw/d). If Tata
Chemicals is unable to provide this information, please provide an
explanation how the following studies can be used to support the GRAS
conclusion:
Howard et al., 1995(a) and (b)
Tsukahara et al., 2003
Correa-Matos et al., 2003
Nakamura et al., 2004
Response: We attempted to calculate (please see below, the last paragraph of this response)
the doses for one of the study. However, as these studies were conducted in weaning animals,
it is bit difficult to determine the equivalent dose on body weight basis, given the rapid
growth or body weight gain. Hence, we are providing discussion as regards any relevance of
these studies from a safety point of view.
It should be noted that all these studies were conducted to investigate the efficacy of FOS in
piglets (Howard et al., 1995a; Correa-Matos et al., 2003; Tsukahara et al., 2003) or weaning
rats (Howard et al., 1995b) and weaning mice (Howard et al., 1995b, Nakamura et al., 2004).
These studies in weaning pigs, rats, and mice indicate that scFOS is unlikely to cause adverse
effects. In general, the piglet is considered as a surrogate model for human infants. In the
studies using the piglet model, the exposure to scFOS was as follows: diet containing FOS
(10%) ad libitum for 10 days (Tsukahara et al., 2003); 3 g FOS/L for 15 days (Howard et al.,
1995b); and 7.5 g/L in formula for 14 days (Correa-Matos et al., 2003). In these studies, no
adverse effects of scFOS were reported. In additional studies, in mice (drinking water
containing 30 g scFOS/L for 14 days) and rats (drinking water containing 30 g scFOS/L for
14 days) also, no adverse effects were reported. These findings from neonatal animal studies
indicate that proposed use of scFOS in infants is unlikely to cause adverse effects.
In the first study, with two separate experiments, Howard et al. (1995b) investigated the
abilities of soluble dietary fiber (including scFOS) to stimulate Bifidobacteria populations
and promote large intestinal mucosal cell proliferation in rats and mice. In these experiments,
the FOS intake in mice was reported as 0.29 g/day, while in the rats it was reported as 0.51
g/day. The initial weight of mice was provided as 22.3 g while for rat it was 51.7 g. The daily
increase in weight of rat was given as 4.4 g/day, so at the end of experiment (14 days) the
weight will be 51.7 + 61.6 = 113.3 g. Based on the information provided in this publication,
the dose of FOS in mice will be approximately 12.6 g/kg bw/day, while in rats it can range
from 4.5 to 9.85 g/kg bw/day.
FDA Query 22. On p. 44 of the notice, Tata Chemicals states “Based on

information from FDA's GRAS Notice Inventory website as of April 28,
Page 13 of 22
2015, the agency has received three notices on FOS and provided "no
questions" letters to all of the notifiers.” We note that this statement and
the subsequent paragraph are out of date and incorrect. Please provide an
updated paragraph that corrects and updates this information.
Response: Thank you for bringing this to our attention. We are sorry for the oversight, as
this description got inserted from our previous GRAS notice and needed to be corrected. The
corrected paragraph should be as follows:
Based on information from FDA’s GRAS Notice Inventory1 website as of July 9, 2021, the
agency has received six notices on FOS and provided “no questions” letters to all the
notifiers. The details of these notices along with the GRN number, date of closure and FDA’s
letter are provided in the below table. A closely related oligosaccharide, galacto-
oligosaccharide, has also been determined to be GRAS for use in a variety of foods in
thirteen GRAS notifications to the FDA. All these GRAS notifications are available at FDA’s
website on GRAS Notices.
GRN
Substance Date of closure FDA's Letter
No.
797 Fructooligosaccharides Nov 15, 2018 FDA has no questions (in PDF)
Short-chain fructo-
717 Feb 13, 2018 FDA has no questions (in PDF)
oligosaccharides
623 Fructooligosaccharides Aug 1, 2016 FDA has no questions
FDA has no questions (previous GRAS

605 Fructo-oligosaccharides Mar 17, 2016
notice by Tata Chemicals)
Short-chain fructo-
537 Feb 6, 2015 FDA has no questions
oligosaccharides
FDA has no questions (additional

44 Fructooligosaccharide Nov 22, 2000
correspondence available)
FDA Query 23. On p. 58 of the notice, the citation for Tsukahara et al.,
2003 contains a typographical error. Please provide the correct citation.
1
Accessible at:
https://www.cfsanappsexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&sort=GRN_No&order=DES
C&showAll=true&type=basic&search=
Page 14 of 22
Response: Thank you for bringing this to our attentions and we are sorry for the oversight
related to the typographical error. The correct reference is as follows:
Tsukahara, T., Iwasaki, Y., Nakayama, K., Ushida, K. 2003. Stimulation of butyrate
production in the large intestine of weaning piglets by dietary fructooligosaccharides and its
influence on the histological variables of the large intestinal mucosa. J Nutr Sci Vitaminol
(Tokyo) 49:414- 421.
Microbiology
FDA Query 24. On p. 10 of the notice, Tata Chemicals states, “A. pullulans
used in the production of scFOS is non-toxigenic and non-pathogenic…”
Please provide a brief summary discussing the safety of A. pullulans.
Response: As this same microorganism (A. pullulans) that was used in the manufacturing of
scFOS in our previous GRAS notice (GRN 605), we did not further elaborate on this.
However, we provide a brief summary of A. pullulans below.
A. pullulans, used in the production of scFOS is registered with the Microbial Type Culture
Collection and Gene Bank (MTCC) under the number MTCC 5490. The characteristics of A.
pullulans, as well as the development, safety, and identity of the production strain has been
established. The production strain, A. pullulans MTCC 5490, was subjected to genetic
identification by 16S ribosomal RNA gene, partial sequence for confirmation. A. pullulans
strain MTCC 5490 is maintained in the Microbial Type Culture Collection and Gene Bank.
The phylogenetic tree based on 16S rRNA and as compared to other related species and
designates was developed for A. pullulans.
A. pullulans is a common black saprobic mould with a world-wide distribution in both indoor
and outdoor environments. It can be found in lake water, on leaves and wood, as well as in
used cosmetics and on foods such as fruits, cereals, tomatoes, and cheese. In the food
industry, A. pullulans is used in the production of food ingredients, including pullulan (GRN
99), beta-glucan (GRN 309). The fungus contains multiple life forms (polymorphic)
including blastospores, hyphae, chlamydospores, and swollen cells. The chlamydospores and
swollen cells are considered resting forms. The fungus produces a green melanin which turns
black over time.
Early clinical studies either failed to establish a pathogenic association or the taxonomic
procedures failed to distinguish their isolates from Exophialia spp. In the past several
decades there have been a few additional reports (Salkin et al., 1986) on the pathogenicity of
A. pullulans for seriously immunocompromised patients, a phenomenon that is considered
possible for most fungi including the baker's yeast Saccharomyces cerevisiae. Indeed there
are far more reports associating this beneficial and safe industrial yeast with various disease
syndromes than the rare associations indicated for A. pullulans. In another case report,
Hawkes et al. (2005) reported a case of A. pullulans fungemia with invasive infection in an
Page 15 of 22
infant. The authors reviewed the previously reported 23 cases of human infection from the
literature (1966-2003). This case in an infant is also, the first case of documented invasive
pulmonary infection and the first patient with a recently repaired cardiac lesion as the
identified risk factor.
Host debilitation is by far the primary factor in the opportunistic or adventitious involvement
of saprobic fungi with humans. Nevertheless, the available evidence for the past three
decades with yeasts and moulds in environmental, industrial and clinical settings, the
involvement of A. pullulans with any adverse human health related problems is extremely
rare.
Based on above, A. pullulans used in the production of scFOS is considered as non-toxigenic
and non-pathogenic.
FDA Query 25. In Table 1 on p. 9 of the notice, Tata Chemicals provides

sampling specifications for Salmonella spp. and Cronobacter sakazakii (C.
sakazakii).
a) Please state whether Tata Chemicals is analyzing multiple 25 g
samples of product or one 100 g sample for Salmonella spp. We
recommend that Salmonella testing be performed on sample sizes no
larger than 25 g to prevent the possibility of false negatives, unless
the method used is validated for larger samples. If analysis is
performed on a sample size larger than 25 g, please discuss the
method and how it was validated.
Response: Please note that Salmonella spp. has been analyzed on multiple 25 g samples
(25 g x 4) and not as single 100 g sample. Sorry for our oversight in not mentioning this.
b) The notice cites method ISO 22964: 2017 for C. sakazakii as “absent
300g.” We note a discrepancy in that this method is validated for test
sample sizes of 10 g. Please clarify this discrepancy and state whether
Tata Chemicals is analyzing multiple 10 g samples of product or one
300 g sample for C. sakazakii. We recommend that C. sakazakii testing
be performed on sample sizes no larger than 10 g to prevent the
possibility of false negatives, unless the method used is validated for
larger samples. If analysis is performed on a sample size larger than 10
g, please discuss the method and how it was validated.
Response: Please note that Cronobacter sakazakii has been analyzed as multiples of 50 g
(50 g x 6) and method was validated using in cerelac matrix (by SGS India). For the
future batches, we will adopt the testing methodology with sample size no larger than 10
g.
Page 16 of 22
FDA Query 26. Please state whether any of the raw materials used in the
fermentation process are major allergens or are derived from major
allergens. If any of the raw materials used are major allergens or derived
from major allergens, please discuss why these materials do not pose a
safety concern.
Response: The raw materials used in the fermentation and scFOS production neither fall
under the major allergen category nor are they derived from major allergens.
We hope the above information and clarification addresses your queries. If you have any
questions or need additional explanation, please let me know.
Thank you for the opportunity to provide this explanation to the agency queries.
Best regards
Madhu Soni, PhD
Agent for: Tata Chemicals Limited, India
Page 17 of 22
References
Demmers, T.A., Jones, P.J., Wang, Y., Krug, S., Creutzinger, V., Heubi, J.E., 2005. Effects
of early cholesterol intake on cholesterol biosynthesis and plasma lipids among infants until
18 months of age. Pediatrics 115(6):1594-1601.
FDA, 2007. Frucrooligosaccharide, GRAS. Agency response additional correspondence
letter: GRAS notice no. GRN 000044. June 1, 2007. Available at: https://wayback.archive-
it.org/7993/20171031035213/https://www.fda.gov/Food/IngredientsPackagingLabeling/GRA
S/NoticeInventory/ucm154400.htm
FSSAI, 2020. Food Safety and Standard Authority of India. Food Safety Standards
(Contaminants, Toxins and Residues) Regulations 2011. Available at:
https://fssai.gov.in/upload/uploadfiles/files/Compendium_Contaminants_Regulations_20_08
_2020.pdf
Friedman, G., Goldberg, S.J., 1975. Concurrent and subsequent serum cholesterol of breast-
and formula-fed infants. Am J Clin Nutr. 28(1):42-45.
Hawkes, M., Rennie, R., Sand, C., Vaudry, W., 2005. Aureobasidium pullulans infection:
fungemia in an infant and a review of human case. Diagn. Microbiol. Infect. Dis. 51:209-213.
Jain, M., Gote, M., Dubay, A.K., Narayanan, S., Krishnappa, H., Kumar, DP. S., Ravi, G.S.,
Vijaysarthi, S.K., Shankar, S., 2019. Safety evaluation of fructooligosaccharide
(FOSSENCE™): Acute, 14-day, and subchronic oral toxicity study in Wistar rats.
Toxioclogy Research Application 2:1-20.
Owen, C.G., Whincup, P.H., Odoki, K., Gilg, J.A., Cook, D.G., 2002. Infant feeding and
blood cholesterol: a study in adolescents and a systematic review. Pediatrics 110(3):597–608.
Salkin, I. F., Martinez, J. A., Kemna, M. E., 1986. Opportunistic infection of the spleen
caused by Aureobasidium pullulans. J. Clin. Microbiol. 23(5):828-831.
Wong, W.W., Hachey, D.L., Insull, W., Opekun, A.R., Klein, P.D., 1993. Effect of dietary
cholesterol on cholesterol synthesis in breast-fed and formula-fed infants. J Lipid Res.
34(8):1403-1411.
Yamamota, Y., A. Yonekubo. 1993. A Survey of Physical Growth, Nutritional intake, Fecal
Properties and Morbidity of Infants as Related to Feeding Methods. “Japanese Infant
Formula Survey.” Central Research Laboratories, Meiji Milk Products Co., Ltd. (Cited in
GRN 44).
Page 18 of 22
Appendix I
Table 1. FDA query 1 - GRN citations and list with correct links
GRN No.;
Page No.
notifier name Correct FDA response letter
in GRAS Correct Notifier reference link
and year; FDA link
notice
response year
Web-link not correct- correct link is as
follows:
GRN 537 http://wayback.archive-
Page 4 (Ingredion, NA (Not applicable) it.org/7993/20171031055001/https://www.
2014) fda.gov/downloads/Food/IngredientsPacka
gingLabeling/GRAS/NoticeInventory/ucm
422895.pdf
GRN 797 follows:
Page 4 NA (Not applicable)
(NFBC, 2018) https://www.fda.gov/media/132054/downl
oad
GRN 797
Page 9 NA (Not applicable) Correct link provided above for Page 4.
(NFBC, 2018)
GRN 537
Page 9 (Ingredion, NA (Not applicable) Correct link provided above for Page 4.
2014)
follows: http://wayback.archive-
GRN 44 (GTC it.org/7993/20171031055001/https://www.
Nutrition 2000) fda.gov/downloads/Food/IngredientsPacka
261587.pdf
https://wayback.archive-
https://www.fda.gov/food/gras-
it.org/7993/20190208035755/https:/www.f
notice-inventory/agency-
Page 10 GRN 605 da.gov/downloads/Food/IngredientsPackag
response-letter-gras-notice-no-
ingLabeling/GRAS/NoticeInventory/ucm4
grn-000605
95918.pdf
GRN 797
(NFBC, 2018)
GRN 537
2014)
Page 18 GRN 537 NA Correct link provided above for Page 4.
http://wayback.archive-
it.org/7993/20171031055001/https://www.
Page 24 GRN 392 NA fda.gov/downloads/Food/IngredientsPacka
277112.pdf
Web-link not correct; correct
link is as follows
https://wayback.archive-
GRN 44 (FDA, it.org/7993/20171031035213/ht
2000) tps://www.fda.gov/Food/Ingred
ientsPackagingLabeling/GRAS/
NoticeInventory/ucm154122.ht
m
Page 19 of 22
Correct web-link is as follows
https://www.cfsanappsexternal.
GRN 537 (FDA, fda.gov/scripts/fdcc/?set=GRA
2015) SNotices&id=537&sort=GRN_
No&order=DESC&startrow=1
&type=basic&search=537
GRN 605 (FDA,
Page 26 Weblink is correct NA (Not applicable)
2016a)
GRN 623 (FDA,
Page 26 Weblink is correct NA (Not applicable)
2016b)
https://www.cfsanappsexternal.fda.gov/scri
GRN 605 (Tata,
Page 26 NA (Not applicable) pts/fdcc/?set=GRASNotices&id=605&sort
2015)
=GRN_No&order=DESC&startrow=1&ty
pe=basic&search=605
GRN 44 (GTC
Page 36 NA (Not applicable) Correct link provided above for page 9
Nutrition, 2000)
GRN 44 (FDA, Correct link provided above for
2000) page 26
2015) page 26
2016a) page 26
2016b) page 26
2017) page 26
2018) page 26
GRN 537
2014)
GRN 717
Page 39 NA (Not applicable) Link is correct
(Galam, 2017)
GRN 979; This
should be 797- NA (correct link provided
Page 39 NA (correct link provided above)
sorry for the above)
typo
NA (correct link provided
Page 39 GRN 623 NA (correct link provided above)
above)
Page 20 of 22
GRN 605 (Tata,
Page 39 NA (Not applicable) Correct link provided above for page 26
2015)
above)
above)
above)
above)
GRN 797
(NFBC, 2018)
GRN 537
2014)
GRN 537
2014)
GRN 44 (GTC
Page 44 NA (Not applicable) Correct link provided above for Page 9
Nutrition, 2000)
follows:
GRN 392 https://www.cfsanappsexternal.fda.gov/scri
(Pfizer, 2011) pts/fdcc/?set=GRASNotices&id=392&sort
=GRN_No&order=DESC&startrow=1&ty
pe=basic&search=392
GRN 537
2014)
Available at:
https://wayback.archive- Available at: http://wayback.archive-
GRN 236. This
it.org/7993/20171031035213/ht it.org/7993/20171031055001/https://www.
should be GRN
Page 47 tps://www.fda.gov/Food/Ingred fda.gov/downloads/Food/IngredientsPacka
334- sorry for
ientsPackagingLabeling/GRAS/ gingLabeling/GRAS/NoticeInventory/ucm
the typo
NoticeInventory/ucm233093.ht 269519.pdf
m
Available at:
Page 47 GRN 233 tps://www.fda.gov/Food/Ingred fda.gov/downloads/Food/IngredientsPacka
m
Available at:
m
Available at: Available at: http://wayback.archive-
Page 47 GRN 489 https://wayback.archive- it.org/7993/20171031055001/https://www.
it.org/7993/20171031035213/ht fda.gov/downloads/Food/IngredientsPacka
Page 21 of 22
tps://www.fda.gov/Food/Ingred gingLabeling/GRAS/NoticeInventory/ucm
ientsPackagingLabeling/GRAS/ 381400.pdf
m
Available at:
m
Available at:
m
Available at:
m
For additional uses- available
at: https://wayback.archive-
it.org/7993/20171031035213/ht
Page 48 GRN 44 tps://www.fda.gov/Food/Ingred NA
ientsPackagingLabeling/GRAS/
m
Page 48 GRN 537 NA Correct link provided above for page 4
Page 48 NA
2000) page 26
Page 48 NA
2016a) page 26
Page 48 NA
2016b) page 26
Page 48 NA
2017) page 26
Page 48 NA
2018) page 26
Correct link provided above for
Page 52 GRN 537 NA
page 26
Correct link provided above for
Page 52 GRN 797 NA
page 26
NA=Not applicable; please note for some GRNs both the “FDA has no questions” letter link as well as link to
full GRAS notice submitted by Notifier is provided.
Page 22 of 22
From: Madhu Soni
To: Morissette, Rachel
Subject: [EXTERNAL] RE: questions for GRN 000990 to be addressed
Date: Wednesday, September 1, 2021 4:15:14 PM
Attachments: image003.png
image007.png
image011.png
image014.png
image016.png
image018.png
scFOS GRAS infant formula-GRN 990-FDA Query-2 responses final.pdf

As per your below email, please find attached responses to FDA queries for GRN 990. If you
need any further clarification please let me know.
Thank you for this opportunity.
Best regards,
Madhu
------------------------------------------
Madhu Soni, PhD, FACN, FATS
Soni & Associates Inc
749 46th Square
Phone: +1-772-299-0746
Cell: +1-772-538-0104
www.soniassociatesnet

Sent: Thursday, August 26, 2021 10:28 AM
Subject: questions for GRN 000990 to be addressed
Dear Dr. Soni,
Below are some additional questions to be addressed for GRN 000990. Please provide your
responses within 5 business days or let me know if you have any further questions.
1. Thank you for your response to our question 15 in the July 19, 2021 amendment.
We note the totality of the available clinical evidence to support the current
GRAS conclusion. However, please state which clinical studies are
critical/pivotal to your GRAS conclusion for scFOS.
2. In the response to question 18 in the July 19, 2021 amendment, Tata Chemicals
states, “Please note that all relevant data and safety studies mentioned in all
these GRAS notices and critical to the GRAS conclusion for scFOS in infant
formula for term infants are appropriately described in our GRAS notice (GRN
000990). As per our understanding, there are no additional studies or
information in these previous GRAS notices that is not described in our GRAS
notice.” However, Table 4 (Studies of Fructans in Infants) in GRN 000537
appears to contain information and studies not discussed in the current notice
(e.g., Kim et al., 2007,[1] Moore et al., 2003[2]). Additionally, GRN 000537
contains an extensive discussion of EFSA’s conclusion on FOS. In this opinion
EFSA stated, in part, “There was an increased prevalence of adverse effects,
including loose stools, in infants fed formula with added
fructooligosaccharides.”[3] A similar discussion of EFSA’s opinion on FOS is not
included in the current safety narrative for GRN 000990.
a. Please provide an explanation how Tata Chemicals can reach a GRAS

conclusion for scFOS in infant formula without information discussed in
previous GRAS notices for scFOS.
b. If Tata Chemicals is intending to incorporate information from previous GRAS

notices into the current notice, please provide the following information for
each study and/or any information being incorporated into the notice: a brief
discussion of the specific data/information being incorporated, the GRN
number that contains the referenced information, and the page numbers in the
GRN where the referenced information can be found. If Tata Chemicals does
not intend to incorporate any information into the notice, please provide
revised statements as identified in question 18 (i.e., on pages 26, 39, and 40 of
the notice) clarifying that no information is being incorporated.
3. In the response to question 21 from the July 19, 2021 amendment, Tata Chemicals
states, “However, as these studies were conducted in weaning animals, it is bit [sic]
difficult to determine the equivalent dose on body weight basis, given the rapid
growth or body weight gain.” While we are not asking for any additional scientific
information to support your response to question 21, we do ask that you confirm your
agreement or disagreement with the following statement: Even though actual body
weights may not be provided in the published studies, a dose on a body weight basis is
possible to calculate in weaning piglets (eg., Hanlon and Thorsrud, 2014[4]) and
piglets in a feeding study are often weighed each day to determine the volume of
formula to dispense (e.g., Monaco et al., 2020[5]).
[1]Kim S-H, Lee DH, Meyer D (2007) Supplementation of baby formula with native inulin has a prebiotic
effect in formula-fed babies. Asia Pac J Clin Nutr. 16(1):172-177
2More et al. (2003) Effects of fructo-oligosaccharide-supplemented infant cereal: a double-blind,
randomized trial. Br J Nutr. 90(3):581-587. doi: 10.1079/bjn2003950
3EFSA (2004) Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request
from the Commission relating to the safety and suitability for particular nutritional use by infants of
fructooligosaccharides in infant formulae and follow-on formulae. The EFSA Journal, 31:1-11.
4Hanlon PR and Thorsrud BA (2014) A 3-week pre-clinical study of 2’-fucosullactose in farm piglets. Food
Chem Toxicol., 74:343-348. doi: 10.1016/j.fct.2014.10.025
5Monaco et al. (2020) Evaluation of 6’-sialyllactose sodium salt supplementation to
formula on growth and clinical parameters in neonatal piglets. Nutrients 12(4):1030. doi:
10.3390/nu12041030
Best regards,
Rachel
-------------------------------------------------------------

tffll U.S. FOOD & DRUG

- ADMINI STRATION
[1] Kim S-H, Lee DH, Meyer D (2007) Supplementation of baby formula with native inulin has a prebiotic
[2] More et al. (2003) Effects of fructo-oligosaccharide-supplemented infant cereal: a double-blind,
[3] EFSA (2004) Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request
[4] Hanlon PR and Thorsrud BA (2014) A 3-week pre-clinical study of 2’-fucosullactose in farm piglets.
Food Chem Toxicol., 74:343-348. doi: 10.1016/j.fct.2014.10.025
[5] Monaco et al. (2020) Evaluation of 6’-sialyllactose sodium salt supplementation to formula on growth
and clinical parameters in neonatal piglets. Nutrients 12(4):1030. doi: 10.3390/nu12041030
RE: Additional Queries for GRN 990 (Short-chain fructooligosaccharide

GRAS notice)
This responds to your email of August 26, 2021 regarding additional
clarifications required for Tata Chemicals’s short-chain fructooligosaccharides
(scFOS) GRAS notice (GRN 000990) for use as an ingredient in infant formulas
for term infants. We are providing a point-by-point response to your queries along
with some relevant clarifications/discussion.
FDA Query 1: Thank you for your response to our question 15 in the July 19, 2021
amendment. We note the totality of the available clinical evidence to support the current
GRAS conclusion. However, please state which clinical studies are critical/pivotal to
your GRAS conclusion for scFOS.
Response: We consider the following two studies as being critical to the GRAS
conclusion for scFOS: Ripoll et al. (2015) and Paineau et al. (2014)
FDA query 2: In the response to question 18 in the July 19, 2021 amendment, Tata
Chemicals states, “Please note that all relevant data and safety studies mentioned in all
these GRAS notices and critical to the GRAS conclusion for scFOS in infant formula for
term infants are appropriately described in our GRAS notice (GRN 000990). As per our
understanding, there are no additional studies or information in these previous GRAS
notices that is not described in our GRAS notice.” However, Table 4 (Studies of
Fructans in Infants) in GRN 000537 appears to contain information and studies not
discussed in the current notice (e.g., Kim et al., 2007,[1] Moore et al., 2003[2]).
Additionally, GRN 000537 contains an extensive discussion of EFSA’s conclusion on
FOS. In this opinion EFSA stated, in part, “There was an increased prevalence of
adverse effects, including loose stools, in infants fed formula with added
fructooligosaccharides.”[3] A similar discussion of EFSA’s opinion on FOS is not included
in the current safety narrative for GRN 000990.
a. Please provide an explanation how Tata Chemicals can reach a GRAS conclusion
for scFOS in infant formula without information discussed in previous GRAS
notices for scFOS.
[1] Kim S-H, Lee DH, Meyer D (2007) Supplementation of baby formula with native inulin has a prebiotic
[2] More et al. (2003) Effects of fructo-oligosaccharide-supplemented infant cereal: a double-blind,

[3]
EFSA (2004) Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request
Page 1 of 6
Response: Sorry for missing the studies by Kim et al. (2007) and Moore et al.
(2003), as well as the discussion of EFSA’s conclusion provided in the
previous GRAS notice GRN 000537. We agree to the need for discussing
these studies as well as the EFSA’s conclusion, for the GRAS determination
of scFOS. Thank you for bringing this to our attention. We are incorporating
Kim et al. (2007) study described on pages 50, 51 and 67 of the GRN 000537
and the Moore et al. (2003) study described on page 46 and 68 of the GRN
000537. We are also incorporating the discussion of EFSA’s opinion on FOS
described in GRN 000537 given on pages 205 and 206. Please note that some
of this information and discussion from these publications is further described
below.
b. If Tata Chemicals is intending to incorporate information from previous GRAS

notices into the current notice, please provide the following information for each
study and/or any information being incorporated into the notice: a brief discussion
of the specific data/information being incorporated, the GRN number that contains
the referenced information, and the page numbers in the GRN where the
referenced information can be found. If Tata Chemicals does not intend to
incorporate any information into the notice, please provide revised statements as
identified in question 18 (i.e., on pages 26, 39, and 40 of the notice) clarifying that
no information is being incorporated.
Response: As indicated above we intend to incorporate information from

previous GRAS notices, particularly GRN 000537, in GRN 000990. For the
studies by Kim et al. (2007) and Moore et al. (2003), as well as for EFSA’s
conclusion, a brief discussion is as follows:
Kim et al. (2007): In this prospective, randomized, double-blind, crossover

study, the effects of chicory inulin on the gut microbiome, the frequency of
defecation, and the pH and consistency of feces was investigated. In this
study, 14 healthy term infants averaging 12.4±6.4 weeks of age were
randomly assigned to receive control formula for 3 weeks, followed by inulin-
supplemented formula, or the 2 treatments in reverse order. Inulin from
chicory roots was added to the experimental formula at a concentration of
1.5±0.3 g/100 g powder (~200 mg/100 ml hydrated formula). The defecation
frequency and amount and consistency of stools daily were recorded.
Anthropometric measures were recorded and fecal samples were collected at
the end of each feeding period. The mean intake of inulin was 1.5 g/day. No
Page 2 of 6
infants were withdrawn from the study, no formula-related adverse effects
were observed, and there were no differences in growth between control and
inulin feeding periods. Stool characteristics during inulin feeding apparently
changed in the expected directions (toward increased frequency, softer stools,
and lower pH) but none of the changes reached statistical significance. There
were no differences between control and inulin feeding in total anaerobic
bacteria or bacteroides, but both bifidobacteria and lactobacilli increased
significantly during inulin intake periods. The investigators concluded that the
addition of native inulin to infant formula elicits a prebiotic response.
The above information is incorporated from GRN 000537 described on pages
50, 51 and 67.
Moore et al. (2003): In this randomized, double-blind, placebo-controlled, 28-

day trial, gastrointestinal (GI) effects of FOS-supplemented infant cereal were
investigated. In this study, 56 healthy term infants aged 4-11 months with
demonstrated tolerance for rice cereal and milk-based formula were assigned
to receive infant cereal with 750 mg of either FOS (n=27) or
maltodextrin/serving (n=29). A daily diary recording of cereal intake, stooling
pattern and characteristics, flatulence, vomiting, spitting up, crying, and
abdominal cramping, was maintained by parents. Anthropometric measures
were taken at baseline and study completion. One infant receiving the FOS-
supplemented cereal and 4 infants receiving the placebo dropped out. The
mean daily consumption of FOS was 740 mg/day and was as high as 3000
mg/day. There were no tolerance issues with the FOS-supplemented cereal,
and infants receiving FOS had more significantly frequent stools with more
regular and softer consistency as compared to those receiving placebo. No
serious adverse events were reported, but 17 infants in the experimental group
had 24 reported nonserious events as compared to 16 infants with 21 non-
serious events in the placebo group. No adverse event was regarded as being
related to the intake of FOS. There were no differences between the 2 groups
in growth. The investigators stated that the present study is one of few studies
documenting tolerance to increased fiber intake in the form of FOS as part of
a weaning food.
The above information is incorporated from GRN 000537 described on pages
46 and 68.
Discussion of EFSA’s opinion: European Food Safety Authority (EFSA,

2004) reviewed an application for the use of only 300 mg oligofructose/100
Page 3 of 6
ml and concluded (described in GRN 000537) that: 1. “There was an
increased prevalence of adverse effects, including loose stools, in infants fed
formula with added fructooligosaccharides.” 2. “As no measures were made
to demonstrate satisfactory water balance, the possibility of increased risk of
dehydration cannot be excluded, raising concerns with respect to the safety of
such formulae.” 3. “There is no evidence of benefits to infants from the
addition of fructooligosaccharides to infant formula at the conditions
specified by the manufacturer while there are reasons for safety concerns.”
In GRN 000537 (pages 205, 206 and 207), the concern expressed by EFSA
for water balance has been extensively discussed and addressed, while two
other aspects of the above conclusion are less central to evaluating the safety
of the intended addition of scFOS to infant formula. The increased prevalence
of “loose stools” is regarded as a beneficial effect of the formula
supplemented with fructans in that the infants receiving these formulas
exhibited stooling performance more closely matching that of breastfed
infants than did infants receiving control formulas without fructans.
As described in GRN 000537, it appears that EFSA (2004) has interpreted
loose, poorly formed, or watery stools as reported by a parent as being
equivalent to clinically diagnosed diarrhea. Generally, diarrhea would indeed
be properly regarded as an adverse effect. However, it has not been reported
in any of the many controlled studies of ingestion of fructans-supplemented
formula by infants (or studies of formula supplemented with GOS or other
oligosaccharides). In the study by Yao et al. (2010), stool composition and
consistency was a primary outcome measure of feeding infant formula
supplemented with 300 or 500 mg oligofructose/100 ml for 8 weeks. No
infant consuming these formulas was reported as having diarrhea, and even
the incidence of parentally reported watery stools was not increased by the
addition of oligofructose, indicating that there was no increase in water loss.
Additionally, the absence of statistically significant long-term benefit in
short-term studies of scFOS or other fructans added at an average of 300
mg/100 ml (compared with an oligosaccharide content of about 800-1200
mg/100 ml in human milk) does not bear upon the safety of the formula and is
not relevant to a determination of whether the intended use is GRAS. Few of
these studies were designed or powered to detect long-term beneficial effects
from the tested interventions.
Page 4 of 6
Furthermore, in GRN 000537 (pages 206, 207), extensive discussion on the
EFSA (2004) considerations of parental classification of stool consistency in
the studies reviewed has been provided. This discussion, along with other
studies and the Food Standards Australia New Zealand (FSANZ, 2008)
critical evaluation of concerns expressed by EFSA relating to water balance,
suggest that the intended use of scFOS by Tata Chemicals is unlikely to cause
adverse effects. The 2008 conclusions of FSANZ regarding the safety of
inulin-supplemented infant formula were repeated in this authoritative body’s
subsequent conclusion that infant formula supplemented with scFOS is
equally safe (FSANZ, 2013).
FDA query 3: In the response to question 21 from the July 19, 2021 amendment, Tata
Chemicals states, “However, as these studies were conducted in weaning animals, it is
bit [sic] difficult to determine the equivalent dose on body weight basis, given the rapid
growth or body weight gain.” While we are not asking for any additional scientific
information to support your response to question 21, we do ask that you confirm your
agreement or disagreement with the following statement: Even though actual body
weights may not be provided in the published studies, a dose on a body weight basis is
possible to calculate in weaning piglets (eg., Hanlon and Thorsrud, 2014[4]) and piglets
in a feeding study are often weighed each day to determine the volume of formula to
dispense (e.g., Monaco et al., 2020[5]).
Response: Sorry for the confusion. We do confirm our agreement with the
statement, “Even though actual body weights may not be provided in the published
studies, a dose on a body weight basis is possible to calculate in weaning piglets
(eg., Hanlon and Thorsrud, 2014) and piglets in a feeding study are often weighed
each day to determine the volume of formula to dispense (e.g., Monaco et al.,
2020).”
We hope the above information and clarification addresses agency queries. If you
have any questions or need additional explanation, please let me know.
Thank you for the opportunity to provide this explanation to the agency queries.
Best regards
Madhu Soni, PhD
Agent for: Tata Chemicals Limited, India
[4]
Hanlon PR and Thorsrud BA (2014) A 3-week pre-clinical study of 2’-fucosullactose in farm piglets.
Food Chem Toxicol., 74:343-348. doi: 10.1016/j.fct.2014.10.025
[5] Monaco et al. (2020) Evaluation of 6’-sialyllactose sodium salt supplementation to formula on growth
and clinical parameters in neonatal piglets. Nutrients 12(4):1030. doi: 10.3390/nu12041030
Page 5 of 6
References:
FSANZ, 2008. Food Standards Australia New Zealand. Final assessment report:
proposal P306, addition of inulin/FOS & GOS to food. July 16.
FSANZ, 2013. Food Standards Australia New Zealand. Approval report-

application A1055, short chain fructo-oligosaccharides. May 23.
Yao et al. (2010). High 2- palmitate and oligofructose in lower protein alpha-
lactalbumin-enriched term infant formula: effects on stool characteristics and stool
composition. J Pediatr Gastroenterol Nutr 50(Suppl 2):E207-208.
Page 6 of 6

GRAS Notice GRN 990 Short Chain Fructooligosaccharides W Amendments

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GRAS Notice GRN 990 Short Chain Fructooligosaccharides W Amendments

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GRAS Notice (GRN) No.

749 46th Square

January 15, 2021

Paulette M. Gaynor, Ph.D.

Subject: GRAS Notification for use of short-chain Fructooligosaccharides in Infant

Dear Dr. Gaynor:

We respectfully submit the attached GRAS notice, on behalf of Tata Chemicals

Enclosure: Three copies of the GRAS notification

1. Part I-SIGNED STATEMENTS AND CERTIFICATION ............................................... 4

1.1. Name and Address of Notifier ...................................................................................... 4

2. Part II - IDENTITY, SPECIFICATION, MANUFACTURING AND TECHNICAL

2.1. Identity ............................................................................................................................ 7

3. Part III - DIETARY EXPOSURE ........................................................................................ 14

3.1. Intended Use Levels and Food Categories ................................................................. 14

4. Part IV - SELF LIMITING LEVELS OF USE ................................................................... 16

Tata Chemicals Page 2 of63 FOS- IF-GRAS

6. Part VI - NARRATIVE ......................................................................................................... 18

6.1. DATA PERTAINING TO SAFETY .......................................................................... 18

6.1.1. Pivotal or Primary Published Clinical Studies of FOS in Infant ............................ 18

7. Part VII- SUPPORTING DATA AND INFORMATION ................................................. 53

Tata Chemicals Page 3 of63 FOS- IF-GRAS

In accordance with 21 CFR § 170 Subpart E consisting of § 170.203 through § 170.285,

Tata Chemicals Page 4 of63 FOS- IF-GRAS

Tel: +91- 020 66549772

Tel: +91- 8976056249

Tata Chemicals Page 5 of63 FOS- IF-GRAS

Tata Chemicals Page 6 of63 FOS- IF-GRAS

2.1.2. Synonyms and Trade Names

FOS; Oligofructose; short-chain fructo-oligosaccharides (scFOS or FOS); Neosugar. The

2.1.3. Chemical Abstract Registry Number

The CAS Registry Number for fructo-oligosaccharides (FOS) is 308066-66-2.

2.1.4. Chemical Formula and Molecular Weight

2.1.5. Chemical Structure

scFOS are a mixture of oligosaccharides consisting of a sucrose molecule (glucose -

Tata Chemicals Page 7 of63 FOS- IF-GRAS

2.1.6. Other Chemically Related Constituents

Tata Chemicals Page 8 of63 FOS- IF-GRAS

Tata Chemicals Page 9 of63 FOS- IF-GRAS

2.3. Manufacturing Process

Tata Chemicals Page 10 of63 FOS- IF-GRAS

Tata Chemicals Page 11 of63 FOS- IF-GRAS

Tata Chemicals Page 12 of63 FOS- IF-GRAS

Process Flow Diagram - Downstream Process -

Activated Carbon Filtration & Polishing Filter Resin Treatment

Liquid Product Evaporation & Polishing Resin & >--. Chromatography

Packaging (HDPE) Spray Drying &

Tata Chemicals Page 13 of63 FOS- IF-GRAS

3.1. Intended Use Levels and Food Categories

3.1.1. Estimated Daily Intake from the Proposed Uses

Tata Chemicals Page 14 of63 FOS- IF-GRAS

Tata Chemicals Page 15 of63 FOS- IF-GRAS

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Table 2. Pivotal or Primary Studies of scFOS in Infants

Dose, Duration Study Design,

Tata Chemicals Page 19 of63 FOS- IF-GRAS

In a parallel feeding randomized, double-blind, 28-day trial in healthy term newborn

Tata Chemicals Page 20 of63 FOS- IF-GRAS

Tata Chemicals Page 21 of63 FOS- IF-GRAS

Tata Chemicals Page 22 of63 FOS- IF-GRAS

Tata Chemicals Page 23 of63 FOS- IF-GRAS

Tata Chemicals Page 24 of63 FOS- IF-GRAS