GRAS Notice (GRN) No.

978
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: sonim@bellsouth.net

November 5, 2020

Food and Drug Administration

Center for Food Safety and Applied Nutrition
Office of Food Additive Safety (HFS-200)
5100 Campus Drive
College Park, MD 20740

Sub_ject: GRAS Notification for Aqueous Olive Pulp Extract {Hidrox®)

Dear Sir/Madam:

In accordance with 21 CFR part 170, subpart E, Oliphenol LLC, through Soni &
Associates Inc. as its agent, hereby submits the enclosed notice of a claim that the food
ingredient Aqueous Olive Pulp Extract (Hidrox®) described in the enclosed notification
dossier is exempt from the premarket approval requirement of the Federal Food, Drug,
and Cosmetic Act because it has been determined to be Generally Recognized As Safe
(GRAS), based on scientific procedures.

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@bellsouth.net

Enclosure: Three copies

www .soniassociates.net
EVALUATION OF THE GENERALLY RECOGNIZED AS SAFE
(GRAS) STATUS OF
AQUEOUS OLIVE PULP EXTRACT (HIDROX®)
AS A FOOD INGREDIENT

Prepared for:
Oliphenol LLC.
26225 Eden Landing Road, Suite C
Hayward, CA 94545

Prepared by:
Soni & Associates Inc.
749 46th Square
Vero Beach, FL 32968

Panel Members
Robert L. Martin., Ph.D.
John A. Thomas, Ph.D., F.A.C.T., F.A.T.S.
Madhusudan G. Soni, Ph.D., F.A.C.N., F.A.T.S.

October, 2020
 EVALUATION OF THE GENERALLY RECOGNIZED AS SAFE (GRAS)
STATUS OF AQUEOUS OLIVE PULP EXTRACT (HIDROX®) AS A FOOD
INGREDIENT

TABLE OF CONTENTS

Part I- SIGNED STATEMENT AND CERTIFICATION .....................................................4

1.1. Basis of Conclusion ....................................................................................................4

1.2. Name and address of organization: ..........................................................................4
1.3. Name of substance: ....................................................................................................4
1.4. Intended conditions of use of Aqueous Olive Pulp Extract (Hidrox®):................4
1.5. Statutory Basis for GRAS conclusion: .....................................................................4
1.6. Exemption from Premarket approval requirements: .............................................4
1.7. Availability of data and information: ....................................................................... 5
1.8. Data exempt from Disclosure: ..................................................................................5
1.9. Certification: ...............................................................................................................5
1.10. Name, position/title of responsible person who signs dossier and signature: .......5
1.11. FSIS/USDA- Use in Meat and/or Poultry: .............................................................5

Part II- IDENTITY AND TECHNICAL INFORMATION ...................................................6

2.1. Description ..................................................................................................................6

2.2. Botanical identification ..............................................................................................6
2.3. Specifications ..............................................................................................................7
2.3.1. Hydroxytyrosol ...........................................................................................................8
2.4. Manufacturing Process ............................................................................................10
2.5. Biologically Active Constituents of Olive ...............................................................12

Part III- DIETARY EXPOSURE............................................................................................15

3.1. Proposed Use Levels and Food Categories ............................................................15

3.2. Estimated Daily Intake ............................................................................................15
3.3. Exposure from Presence in Food ............................................................................17

Part IV- SELF LIMITING LEVELS OF USE ......................................................................19

Part V- EXPERIENCE BASED ON COMMON USE IN FOOD BEFORE 1958 .............20

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Part VI- NARRATIVE .............................................................................................................21

6.1. Traditional and Current Uses .................................................................................21

6.2. Data Pertaining to Safety.........................................................................................21
6.2.1. Pivotal Studies of Aqueous Olive Pulp Extract .....................................................21
6.2.1.1 Subchronic Toxicity Study of Aqueous Olive Pulp Extract .................................23
6.2.1.2 Developmental and Reproductive Toxicity of Aqueous Olive Pulp Extract ......24
6.2.1.3 Genotoxicity Studies of Aqueous Olive Pulp Extract ...........................................26
6.2.1.3.1 Bacterial Reverse Mutation Assay (Ames test) .....................................................26
6.2.1.3.2 Chromosomal Aberrations Assay ...........................................................................27
6.2.1.3.3 Micronucleus Assay .................................................................................................27
6.2.1.4 Acute and Short-term Toxicity Studies of Aqueous Olive Pulp Extract ............28
6.2.1.5 Absorption Study of Aqueous Olive Pulp Extract ................................................29
6.2.1.6 Human Study of Aqueous Olive Pulp Extract.......................................................29
6.2.2. Secondary Pivotal Published Studies of Olive Extracts........................................29
6.2.2.1. Subchronic Toxicity Studies ....................................................................................30
6.2.2.2. Mutagenicity and Genotoxicity Studies .................................................................32
6.2.2.3. Acute Toxicity Study................................................................................................34
6.2.2.4. Absorption, Distribution, Metabolism and Excretion ..........................................34
6.2.2.5. Human Studies with Olive Preparations ...............................................................36
6.2.3. Secondary Unpublished Studies of Olive Extracts................................................38
6.2.3.1. Secondary Unpublished Toxicity Studies of Olive Extracts .................................38
6.2.3.1. Secondary Unpublished Human Studies of Olive Extracts..................................39
6.2.4. Corroborative Information .....................................................................................40
6.2.4.1. FDA GRAS Notices on Olive Phenolic Preparation and Hydroxytyrosol..........40
6.2.4.1.1. GRN 726- Phenolic Preparation from Olive Fruit................................................41
6.2.4.1.2. GRN 600 and 876- Hydroxytyrosol ........................................................................42
6.2.4.2. Evaluation by the European Food Safety Authority (EFSA) ..............................43
6.7. Expert Panel Review, Summary and Discussion...................................................43
6.8. Expert Panel Conclusion ..........................t...............................................................47

Part VII- SUPPORTING DOCUMENTS AND REFERENCES.........................................48

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Part I- SIGNED STATEMENT AND CERTIFICATION
I.I. Basis of Conclusion
This GRAS conclusion for the use of Aqueous Olive Pulp Extract (Hidrox®) as a food
ingredient has been reached in accordance with requirements as defined in 21 CFR 170.220.
1.2. Name and address of organization:
Oliphenol LLC.
26225 Eden Landing Road, Suite C
Hayward, CA 94545
USA
Phone: +1-833-654-7436
email: info@Oliphenol.com
1.3. Name of substance:
The name of the substance of this GRAS assessment is Aqueous Olive Pulp Extract (Hidrox®).
1.4. Intended conditions of use of Aqueous Olive Pulp Extract (Hidrox®):
Aqueous Olive Pulp Extract (Hidrox®) is intended to be used as a food ingredient and as an
antioxidant in selected conventional food categories, such as: bakery products; beverages; dairy
products and substitutes; desserts; fats and oils; fruit juices and nectars; dry seasoning mixes for
meat, poultry and fish; chewing gum; sauces, dips, gravies and condiments; snacks; and
vegetable juices to deliver 5 to IO mg of hydroxytyrosol per serving (reference amounts
customarily consumed, 21 CFR IO1.12). The actual amount of Aqueous Olive Pulp Extract
(Hidrox®) added (approximately 150 to 300 mg/serving) to the food categories will be adjusted
such that the resulting addition corresponds to the proposed use levels of hydroxytyrosol. The
intended use of Aqueous Olive Pulp Extract is in the same food products and at the identical
levels mentioned in the GRN 726e1 (Phenolic preparation from olive fmit). It is recognized that
there are Standard of Identity requirements for some of the specified foods and these foods will
not be referred to by their commonly recognized names.
1.5. Statutory Basis for GRAS conclusion:
This GRAS conclusion is based on scientific procedures in accordance with 21 CFR l 70.30(a)
and l 70.30(b).
1.6. Exemption from Premarket approval requirements:
Oliphenol LLC (Oliphenol) has concluded that Aqueous Olive Pulp Extract (Hidrox®) is not
subject to the premarket approval requirements of the Federal Food, Drug, and Cosmetic Act
based on our conclusion that Aqueous Olive Pulp Extract (Hidrox®), meeting the specifications
cited herein, and when used as a food ingredient and as an antioxidant, is GRAS and is therefore
exempt from the premarket approval requirements.
It is also our opinion that other qualified and competent scientists reviewing the same publicly
available toxicological and safety information would reach the same conclusion. Therefore, we

1 Accessible at: https://www.fda.gov/media/l 11692/download, and at: https://www.fda.gov/media/l 11827/download

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have also concluded that Aqueous Olive Pulp Extract (Hidrox®), when used as described in this
dossier, is GRAS based on scientific procedures.
1.7. Availability of data and information:
The data and information that are the basis for this GRAS conclusion will be made available to
FDA upon request by contacting Dr. Crea at the below address. The data and information will be
made available to the FDA in a form in accordance with that requested under 21 CFR
170.225(c)(7)(ii)(A) or 21 CFR I 70.225(c)(7)(ii)(B).

Dr. Roberto Crea

Chief Executive Officer
Oliphenol LLC.
26225 Eden Landing Road, Suite C
Hayward, CA 94545
USA
Phone: +I 833-654-7436
Email: robertocrea@oliphenol .com
1.8. Data exempt from Disclosure:
Part I through Part VII of this GRAS assessment does not contain any privileged or confidential
information such as trade secrets and/or commercial or financial information and can be made
publicly available.
1.9. Certification:
Oliphenol 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 Oliphenol, including any favorable or unfavorable information, and pertinent
to the evaluation of the safety and GRAS status of the use of Aqueous Olive Pulp Extract
(Hidrox®). Oliphenol accepts responsibility for the GRAS conclusion that has been made for
Aqueous Olive Pulp Extract (Hidrox®) as described in this dossier.
1.10. Name, position/title of responsible person who signs dossier and signature:
Dr. Roberto Crea
Chief Executive Officer
Oliphenol LLC.
26225 Eden Landing Road, Suite C
Hayward, CA 94545
r · - ("r__ _
Signature:

1.11. FSIS/USDA- Use in Meat and/or Poultry:

Oliphenol does not intend to add Aqueous Olive Pulp Extract (Hidrox®) to any meat and/or
poultry products that come under USDA jurisdiction. Therefore, 21 CFR 170.270 does not
apply.

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Part II- IDENTITY AND TECHNICAL INFORMATION
2.1. Description
The subject of this GRAS assessment, Aqueous Olive Pulp Extract (Hidrox®) is a
standardized powder and liquid derived from the juice of olives (Olea europaea L.). The
preparations ( powder and liquid) have an odor of processed olives and a characteristic aromatic
sour/olive flavor. The powder is composed of 98-99% dry solids. The extract is prepared by
water extraction of the juice recovered during the milling of olive oil. General descriptive
characteristics of Aqueous Olive Pulp Extract (Hidrox®) are summarized in Table l. The
biologically active constituents of Hidrox® are polyphenols. Among the phenolics, the major
constituent of the pulp extract is hydroxytyrosol ( 50-70%), while other polyphenols present
include oleuropein ( 5-l 0%), tyrosol (0.3%), oleuropein aglycone and gallic acid. All of the
polyphenols present in the extract are also found in olive oil and are thus commonly consumed.

Table 1. General Descriptive Characteristics of Aaueous Olive Pulp Extract (Hidrox®)

Parameter Descriotion (Oliohenol)*
Plant Source Olea
- europaea
-
Olive tree; Oliver; Olivenbaum; Oliva; Olea europea oil;
Other names
Olea europea extract
Part used Fruit
Startin_g material Juice recovered durinQ. the milling of olive oil
Active constituents Phenolic compounds, includin_g hydroxytyrosol
Appearance Dried powder or liquid
Color Golden brown powder; Dark brown, semi-viscous liquid
Odor Processed olives
Flavor Characteristic aromatic sour/olive
Preserve in tight containers and prevent exposure to
Storage
excessive heat
Shelf life 2 vears
*Based on information provided by Oliphenol (2020)

2.2. Botanical identification

The hierarchical classification of the source material, Olea europaea, is presented in
Table 2. The plant is famous for its olive's fruits or "olive," which are the source for a commonly
consumed oil, known as olive oil, around the world and one of the core ingredients in
Mediterranean cuisine. There are thousands of cultivars of the olive. In Italy alone at least three
hundred cultivars have been enumerated, while only a few are grown to a large extent. The olive
tree is an evergreen tree or shrub native to Mediterranean Europe, Asia, and Africa. It is short
and squat, and rarely exceeds 8-15 m in height. The trunk is typically gnarled and twisted. The
leaves are silvery green, oblong, measuring 4-10 cm long and 1-3 cm wide. The small white
flowers, with four-cleft calyx and corolla, two stamens and bifid stigma, are borne generally on
the last year's wood, in racemes springing from the axils of the leaves. The fruit is a small drupe
1-2.5 cm long, thinner-fleshed and smaller in wild plants than in cultivars. A typical picture of
olive branch with fruits is presented in Figure 1.

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 Figure 1. Typical Picture of Olea europaea Plant with Fruits

. I Cl ass1'fi1cafion o f Ofea eurooaea

Ta bl e 2 Taxonom1ca
Rank Scientific Name and Common Name
Kingdom Plantae - Plants
Subkingdom Tracheobionta - Vascular plants
Suoerdivision Spermatophyta - Seed plants
Division Magnoliophyta - Flowering plants
Class Magnoliopsida - Dicotyledons
Subclass Asteridae
Order Scrophulariales
Family Oleaceae - Olive familv
Genus Olea L. - olive
Soecies Olea europaea L. - olive
Source: USDA, Natural Resources Conservation Service

2.3. Specifications
Food grade specifications of Aqueous Olive Pulp Extract (Hidrox®) powder and liquid
by Oliphenol are summarized in Table 3. The polyphenol content of powder is>12%, while that
of liquid is>7.5%. The hydroxytyrosol (Figure 2) content for powder is >3.5% and for liquid is
>3%. Aqueous Olive Pulp Extract is soluble in water (> 95%). Oliphenol intends to market two
different products derived from olives. The comparison of specifications with certificate of
analysis from three non-consecutive lots for Hidrox® Freeze-Dried Powder 12% ( Table 4) and
for three lots ofHidrox® Liquid 1 OX ( Table 5) demonstrate that these products are produced
consistently and meets the food grade specifications.

OH
HO

OH
Figure 2. Chemical Structure of Hydroxytyrosol

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2.3.1. Hydroxytyrosol
The available information suggest that hydroxytyrosol is a major biologically active
constituent of the phenolic fraction of olive extract and olive oil. It is also known as 3,4-
dihydroxytyrosol or 3,4-dihydroxyphenylethanol ( CAS No.: 1 0597-60-1; Figure 2). The
chemical formula of hydroxytyrosol is CsH 1003 and the molecular weight is 154. In addition to
olives, the presence of hydroxytyrosol has also been identified and quantified in wines ( Di
Tommaso et al., 1 998). In olive oil, hydroxytyrosol is found either as the simple phenol or
esterified with elenolic acid to form oleuropein aglycone.Hydroxytyrosol in pure form is a clear,
colorless, tasteless liquid and can be soluble in water or lipid. Although hydroxytyrosol
represents a minor constituent of the aqueous olive pulp extract, as well as of the olive oil
phenolic fraction, it is considered the most potent ( as measured by ORAC) phenolic antioxidant
of Aqueous Olive Pulp Extract and of olive oil. The presence of hydroxytyrosol in olive oil
prevents its autoxidation and, thus significantly contributes to its shelf life.

Table 3. Specifications of Aqueous Olive Pulp Extract

Characteristic/specification
Description II HI DROX® Freeze- HI DROX® Liquid Method
Dried Powder 1 2% I OX
,

Golden brown to Brown to dark

Appearance Visual
purolish oowder purplish
Flavor Sour/Olives Sour/Olives .
Organoleotic
Odor Processed olives Processed olives Organoleptic
pH (in I _g/ 1 0 mL water) 3-4 3-4 AOAC 981.12
Protein (% w/w) 3-8 3-6 AOAC. 925.09
Ash (% wt/wt) 1 2-20 1 2-20 AOAC 942.05
Solubility in water >95 >98 In House
Fat (% w/w) 8- 1 8 8- 1 5 AOAC 954.02
Carbohvdrates (¾w/w) 45-74 30-45 CFR 2 1 -calc.
Moisture (% w/w) <5 34 AOAC. 925.09
Simple
.,__... & Polyphenols (%) > 1 2 >7.5 .
Folin-Ciocalteu (UY)
Hydroxytyrosol >3.5 >3.0
JAOCS. 75/1 1 ( 1 998)
(modified)
.Heavy
. _ metals
Lead (mg/kiz) <0. 1 <0. 1 AOAC 993. 1 4 Mod.
Arsenic ( mg/ke:)
- <0. 1 <0. 1 AOAC 993 . 1 4 Mod.
Cadmium (mg/kg) <0. 1 <0. 1 AOAC 993 . 1 4 Mod.
.
Mercury (mg/kg) <0.01 <0.01 AOAC 993 . 1 4 Mod.
Microbial standards
Colifonns
" and E.coli ( MPN/g) <3 <3 .
USP Chaoter 61
Total Aerobic Plate Count
< 1 000 < 1 000 USP Chapter 62
(CFU/g)
Staohvlococcus aureus ( MPN/g) <3 <3 USP Chaoter 62
Salmonella - (ELFA) presumptive Negative Negative USP Chaoter 62
---
Mold (CFU/g) <10 <10 USP Chaoter 62
Yeast (CFU/Q) <20 <20 USP Chaoter 62
MPN = Most probable number; CFU = Colony fonrnng u111ts

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 Table 4. Specification and Certificate of Analysis for H I DROX® Freeze-Dried Powder 1 2 %
Lot Number
Parameter Specifications
1 2- 1 20928-000 1 2- 1 30 1 1 4-000 1 2-140108-001
Appearance Golden brown to purplish Conforms
Conforms Conforms
POWder
Flavor Sour/Olives Conforms Conforms Conforms
Odor Processed olives Conforms Conforms Conforms
pH (in I g/ I 0mL water) 3-4 3.5 3.4 3.7
Protein (% w/w) 3-8% 3.52% 3.2% 3.86%
Ash (% wt/wt) 1 2-20% 1 3% 1 2.3% 1 2.78%
Solubility in water >95% Conforms Conforms Conforms
Fat (% w/w) 8- 1 8% 1 0.85% 9.00% 8.36%
Carbohydrates (¾w/w) 45-74% 72% 75% 74.59%
Moisture (% w/w) <5% 3.52% 3.2% 3.86%
Simple & Polyphenols > 1 2% 13.78% 1 2.78% 15.93%
Hydroxytyrosol >3.5% 3 .7 1 % 3.55% 4.34%
Oxygen Radical Absorbance
Capacity (ORAC) - µmoles ,::2500 3444 2978 2574
Trolox equivalence (TE)/g
Microbial standards
Coli forms and E.coli <3 <3 <3
<3
(MPN/g)
Total Aerobic Plate Count <IO <10 <10
< 1 000
(CFU/g)
Staphylococcus aureus <3 MPN/g <3 MPN/g <3 MPN/g
<3
(MP N/g )
Salmonella - (ELFA) Negative Negative Negative
Negative
presumptive (oer 25 g)
Mold (CFU/g) < 1 00 <10 < J OO <10
Yeast (CFU/g) <200 <10 < J OO <10
Heavy metals*
Arsenic (ppm) <0.1 0.042 0.033 0.133
Cadmium (ppm) <0.1 <0.005 <0.007 <0.009
Lead (ppm) <0.1 0.033 0.032 0.154
Mercury (ppm) <0.01 <0.007 <0.007 <0.009
*Heavy metal analysis was performed from different lots (Lot # 1 2- 1 70623-000; Lot # 1 2- 1 8 1 206-00 I ; Lot
#12-1 9030 1 -000)

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 Table 5. Certificate of Analysis of HIDROX® Liquid l0X
Lot Number
Parameter Specifications
t ox-1 2 1 1 1 9-000 tox-1 2 1 1 1 9-00 1 t ox-1 2 1 1 19-002
Appearance Dark brown, semi-
Conforms Conforms Conforms
viscous liquid
Flavor Sour/Olives Conforms Conforms Conforms
Odor Processed olives Conforms .
Conforms Conforms
pH (in lg/l0mL water) 3-4 3.1 3.1 3.1
Hydroxytyrosol (g/kg) > 30 34 30 30
Colifom1s and E.coli (MPN/g) <3 <3 <3 <3
Total Aerobic Plate Count
,
< 1 000 <10 <10 <10
.
(CFU/g)
-
Staphylococcus aureus (MPN/g) <3 <3 <3 <3
Salmonella - (ELFA)
Negative Negative Negative Negative
presumptive
,- --- ---(per 25 g)
.
Mold (CFU/g) <1 000 <10 <10 <10
Yeast (CFU/g) < 1 000 <10 <10 <10
Heavy Metals
Lead (mg/kg) <I 0.04 0.036 0.037
-
Arsenic (mg/kg)
Cadmium (mg/kg)
<I 0.081 0.073 0.075
<0.5 0.003 0.003 0.003
'
-
Mercury ( mg/kg) <0.1 0.01 0.01 0.01

2.4. Manufacturing Process

The production process for Aqueous Olive Pulp Extract (Hidrox®) is illustrated in Figure
3. Aqueous Olive Pulp Extract is manufactured according to current good manufacturing
practices ( GMPs). The extract is manufactured at Specialty Concentrates, LLC, 9505 Rd. 301/2,
Madera, CA. 93638, U.S.A. The FDA food facility registration number for the manufacturing
facility is prl 144.
Aqueous Olive Pulp Extract (Hidrox®) is manufactured from byproducts of olive oil
processing. Olives are transported to the process plant in large bins. The olives are transferred
into a receiving hopper and then to a vibrating tray dry cleaner to remove olive leaves, stones,
mud, and dirt. This is followed by passing the olives over a de-stemmer to remove any remaining
stems. Following this step, the olives pass through a rotary washer, vibratory tray, and de­
watering screen for removal of any small remaining debris. The de-watering screen also removes
most of the water from the surface of the olives. Subsequently, the olives are transferred to the
stoner, which presses the olives into a minced pulp, and separates the olive pit from the pulp
without crushing the pit. Alternatively, olives go directly from the washer to the crushing devise
( stone or hammer) where a mix ( slurry) of biomass, oil, emulsified water Uuice) and wood from
pits is generated. Although the manufacturing process hereby described is related to pitted olives,
similar results in producing Aqueous Olive Pulp Extract (Hidrox®) can be expected with olives
that are not pitted.

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 Olive Pomace
I

Three Phase dcanter- Vertical

Centrifugation

I -
Aque<y-1s juice
_,
,.
I
Acidification with 1% citric acid to pH 3.5 - 4.5
and stored in stainless steel 304 tanks (Hidrox)

l
AHer two or more months, hydrolyzed Juice (0.3-
0.5% Total Polyphenols) is filtered through a 0.2µm
membrane tangential filtration unit to obtain a

..
perme4te liquid

The permeate liquid is processed into liquid

HIDROX 2% TP (20 Btix) by vacuum concentrator
•
Collected, weighed, sealed and delivered to
Oliphenol LLC for further repackaging and/or use
in branded products

HIDROX 12 % Dry Powder

~
HIDROX l0X liquid

Liquid HIDROX

Liquid HID ROX

Freeze Dry Processing

Processing to HIDROX I OX (60

Brix) by vacuum concentrator
HIDROX 12% Dry Powder

Collected in drums, QC'ed and Sealed and Collected in drums, QC'ed and Sealed and
delivered to Oliphenol LLC for further delivered to Oliphenol LLC for further
repackaging and/or use in branded products repackaging and/or use in branded products

Figure 3. Manufacturing process of Aqueous Olive Pulp Extract (Hidrox l0X and Hidrox 12%)

The olive pulp is pumped into one of two double sets of warm water-jacketed cut-and­
fold kneaders, where the pulp is gently stirred. The olive pulp is held at a warm temperature (28-
300C) for approximately one hour to facilitate the separation of oil from the aqueous phase.
Following this '"kneading" process, the mixture is transferred into a horizontal centrifuge
(decanter) where three fractions, oil, solid particles (or cake) and vegetation water, are separated.

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Vegetation water, generated from the decanter is collected into a separate reservoir and pumped
into a vertical centrifuge to separate the residual oil and large particulate solids from the
vegetation water. Approximately 90% of the vegetation water is collected into stainless steel
tanks and acidified with food grade citric acid to a specific pH range. The acid-treated olive juice
is stored at room temperature for a minimum of 2 months (and can be up to 6-8 months), where
oleuropein is hydrolyzed to hydroxytyrosol and elenoic acid. This process is monitored by HPLC
until a minimum conversion ratio of 5: I is attained. The liquid is then subjected to filtration
through a 0.2 µm tangential membrane filtration unit to remove residual non soluble particulates
and microorganisms. The resulting hydrolyzed juice 0.3%-0.5% Total Polyphenols (TP) (called
Hidrox® 0.5%) is then concentrated into a semi-viscous liquid using a low-temperature, high­
vacuum evaporation process. This produces a convenient concentration of 2% Total Polyphenols
(TP) (Hidrox 2% or 20 Brix). This liquid can then be used for producing the high concentration
syrup, and/or the production of the freeze-dried powder. In the first case, the concentrated syrup
is standardized to 25 g per kg (2.5%) hydroxytyrosol and is called Hidrox® Liquid ! OX. The
manufacturing flow chart for Hidrox® Liquid I OX is provided below.
For the freeze-dried powder of the Aqueous Olive Pulp Extract, the 2.0% TP liquid
preparation mentioned above (Hidrox® 2.0%) is used to produce a standardized dry product,
called Hidrox 12% TP. This powder product is produced by freeze-drying the Hidrox® 2.0%
product, resulting in a powder consisting of I 0-12% phenolic compounds (Hidrox 12% TP
freeze-dried powder). The manufacturing flow chart is provided in Figure 3.
2.5. Biologically Active Constituents of Olive
Olive fruit is well known for the presence of simple, as well as complex phenolic
substances. The phenolic content and composition of phenols in whole olives varies and depend
on the altitude where the olive trees are grown, the harvesting time and the processing conditions
(Soni et al., 2006). Similarly, the levels of phenolics in olive oil also depend on other factors
such as cultivar, climate, ripeness of olives, preparation and storage of the oil. These phenolics
are responsible for the stability of the oil from oxidation as well as its organoleptic properties
(Visioli and Galli, 200 I ). Phenolic compounds in olive oil are present at levels up to I % by
weight, both as simple (hydroxytyrosol and tyrosol) and complex chemicals (hydroxytyrosol or
tyrosol esterified to elenolic acid, in the form of oleuropein and ligstroside, respectively)
(hydroxytyrosol + elenolic acidt_, oleuropein and tyrosol + elenolic acidt---, ligstroside; Figure 4).
Oleuropein and ligstroside in intact olives are present in the glycosidic, relatively polar form.
During production ( crushing) process, hydroxytyrosol and tyrosol, as well as the lipid soluble
oleuropein and ligstroside aglycones, are partially released (5-10% of the total in olives) from
olives into the oil, while a substantial proportion of these constituents remain in the water phase
(vegetation water).

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 R 0 0

HO
Tyroso//Hydroxytyroso/
0

Elenolic acid
R RI R2
OH H H Oleuropein
H H H Ligstroside
Figure 4. Most relevant phenolics found in olive and olive products.
Hydroxytyrosol is formed by cleavage where indicated

The phenolic content of two brined olive drupe types ( black and green) has been
investigated by Owen et al. ( 2003) ( Table 6). The green olives were found to contain primarily
hydroxytyrosol, while the black olives contain tyrosol, hydroxytyrosol, dihydrocaffeic acid,
dihydro-p-coumaric acid ( phloretic acid), acetoside ( a disaccharide linked to hydroxytyrosol and
caffeic acid), acetoside isomer and the flavonoids apigenin and luteolin. These investigators also
reported that consumption of approximately 50 g of black olive pericarp would provide about
400 mg of phenolic substances to the daily dietary intake, while a similar quantity of extra virgin
olive oil ( produced with conventional methods) provides about 12 mg. In another report on
analysis of 48 olive samples, Romero et al. ( 2004) repo11ed that the 'turning color olives' in brine
had the highest concentration ofpolyphenols ( approximately 0.t12%).

Table 6. Basic Characteristics and Phenolics

in Black vs. Green-brined olives
Olive tvpe
Component
Black Green
-
Pericarp
Total g wet wt 7 1 .78 1 1 1 .60
Total g dry wt 35.89 29.30
G dry wt. oer drupe 1 . 79 1 .46
Water (% of wet wt) 50.00 73.70
Phenolics in pericarp
Mgper drupe 29.43 6.56
% of wet wt 0.82 0.12
% o f dry wt. 1 .64 0.458
Oil
Total g
I-
5.52 1 8.22
% of wet wt. 7.69 1 6.3
% of dry wt. 1 5 .4 62.2
Values for 20 drupes of each olive type

Oliphenol Page 1 3 of 53 Hidrox®-GRAS

 Olive mill water, also called as vegetation water, is a good source of phenolic
antioxidants (1-1.8% w/v), as during the pressing of the drupes approximately 90% of the
phenols in olives are transferred to the water phase. Visioli and Galli (2003) reported that
approximately 10-20% of the total phenol content from vegetation water can be recovered; the
only bioactive catechol recovered is hydroxytyrosol. In another study, Fernandez-Bolanos et al.
(2002) reported extraction of 3 kg of hydroxytyrosol (90-95% purity) from 1000 kg of olives
during liquid-solid waste of two-phase (conventional) olive oil processing.

Oliphenol Page 14 of53 Hidrox®-GRAS

Part III- DIETARY EXPOSURE
3.1. Proposed Use Levels and Food Categories
Oliphenol intends to use Aqueous Olive Pulp Extract (Hidrox®) in 11 food categories:
bakery products; beverages; dairy products and substitutes; desserts; fats and oils; fruit juices and
nectars; dry seasoning mixes for meat, poultry and fish; chewing gum; sauces, dips, gravies and
condiments; snacks; and vegetable juices to deliver 5 to I O mg of hydroxytyrosol per serving
( reference amounts customarily consumed, 21 CFR 101.12). As the subject of this present GRAS
document contains about 3.5% hydroxytyrosol, the per serving use levels of Aqueous Olive Pulp
Extract (Hidrox®) will be approximately 150 to 300 mg/serving. The food serving size to which
the extract will be added corresponds to the gram weight or mL volume of food as specified by
References Amounts Customarily Consumed ( RACCs) for food labeling based on FDA's final
rule, effective July 26, 2016, with the compliance date of July 26, 20 1 8 ( Federal Register, 2016).
Table 7 lists the 11 food categories to which Aqueous Olive Pulp Extract is proposed for use,
descriptions of the types of foods within the category that was included in the assessment, the
serving size associated with each food type, and the maximum use level of the extract.
Aqueous Olive Pulp Extract (Hidrox®) is intended for use in the same foods and at
identical levels of addition to those described in the GRAS notification on "Phenolic preparation
from olive" ( GRN 726) by DSM Nutritional Products, LLC's ( DSM, 2017). The Aqueous Olive
Pulp Extract (Hidrox®) will not be used in any foods for which food standards would preclude
its use. Foods that are intended for infants and toddlers, such as infant formulas or foods
formulated for babies or toddlers, and meat and poultry products that come under USDA
jurisdiction are excluded from the list of intended food uses of Aqueous Olive Pulp Extract
(Hidrox®).
3.2. Estimated Daily Intake
As mentioned above, Aqueous Olive Pulp Extract (Hidrox®) will be used in bakery
products; beverages; dairy products and substitutes; desserts; fats and oils; fruit juices and
nectars; dry seasoning mixes for meat, poultry and fish; chewing gum; sauces, dips, gravies and
condiments; snacks; and vegetable juices to deliver 5 to I O mg of hydroxytyrosol per serving.
The intended use of Aqueous Olive Pulp Extract (Hidrox®) is in the same foods and at identical
levels of addition to those described in GRN 726 by DSM ( 2017). There are no new food uses
proposed for Aqueous Olive Pulp Extract. The application of Aqueous Olive Pulp Extract
(Hidrox®) to the same foods and at the same levels ( 5-10 mg hydroxytyrosol/serving) as those
described in GRN 726 is not expected to noticeably affect the intake of hydroxytyrosol in the
diet of the public from introduction into the market by another supplier who will have to compete
in essentially the same market and in same foods.
In GRN 726, the intake analysis for the US population's consumption of hydroxytyrosol,
from existing and proposed uses was based on food consumption records collected in the What
We Eat in America ( WWEIA) component of the National Health and Nutrition Examination
Surveys ( NHANES) conducted in 2007-2008 and 200 9-2010 ( DSM, 2017). This continuous
survey is a complex multistage probability sample designed to be representative of the civilian
US population. The NHANES datasets provide nationally representative nutrition and health data
and prevalence estimates for nutrition and health status measures in the US. In GRN 726
submitted to the FDA, DSM ( 2017) reported that the cumulative dietary exposure to

Oliphenol Page 15 of 53 Hidrox®-GRAS

 hydroxytyrosol for the total users only US population ( 2 years and older) is 30 mg/person/day
( 0.5 mg/kg bw/day) at the mean and 52 mg/person/day ( 0. 9 mg/kg bw/day) at the 90th percentile.
In a response letter of February 28, 20t1 8 to URN 726, FDA ( 20 1 8) did not question the intake
estimate analysis performed and presented by DSM ( 2017).

Table 7. Proposed Uses of Aqueous Olive Pulp Extract (Hidrox®) in Foods*

Use Level (ml!/servinl!) RACCb
Food Category
Hydroxvtvrosol Hidrox®• (g/serving)
Bakery Products
Crackers that are usually used as snacks 5 1 50 30
Croutons 5 150 7
Grain-based bars with or without filling or coating (e.g., 10 300 40
breakfast bars, granola bars, rice cereal bars)
Protein based, meal replacement and enern.y bars 10 300 40
Beveral!es
Sport drinks, energy drinks, milk-based meal replacements, 5 1 50 240
flavored waters and fruit-flavored drinks
Dairy Products and Substitutes
Yogurt 10 300 225
Desserts
Frozen yogurt 10 300 120
Fats a n d Oils
Butter, margarine, oil and shortening 5 1 50 15
Dressing for salads 5 150 30
Mayonnaise, sandwich spreads, mayonnaise-type dressings 5 150 15
Fruit and Fruit Juices
Fruit juices and fruit nectars 5 1 50 240
Miscellaneous
Meat, poultry, and fish coating mixes, dry; seasoning mixes, dry 5 1 50 4.5
- (e.g., chili seasoning mixes, pasta salad seasoning mixes)"
Chewing gum 10 300 3
Sauces, Dios, Gravies, Condiments
Major main entree sauces (e.g., spaghetti sauce) 5 150 125
Minor main entree sauces (e.g., pizza sauce, pesto sauce), other 5 150 60
sauces used as toppings (e.g. gravy, white sauce, cheese sauce),
cocktail sauce
Major condiments: catsup only 5 1 50 15
- -----------
Barbecue sauce, hollandaise sauce, tartar sauce, other sauces for 5 1 50 30
dipping (e.g., mustard sauce, sweet and sour sauce), all dips
- (e.g., bean dips, dairv-based dios, salsa)
Snacks
All varieties, chips, pretzels, popcorns, extruded snacks, fruit- 5 1 50 30
based snacks ( e.g., fruit chips), grain-based snack mixes
Yel!etable Juices
Vegetable juice 5 1 50 240
* Adapted from GRN 726
, Hidrox contains approximately 3.5% hydroxytyrosol
b RACC= Reference Amounts Customarily Consumed per eating occasion - 2 1 CFR § I O 1 . 1 2 (CFR, 2014). When a range of
values is reported for a particular food-use, particular foods within that food-use may differ with respect to their RACC.
c The estimated RACC for dry seasoning mixes was estimated to be 4.5 g dry spice rub (i.e., 2 teaspoons per serving) based
upon publicly available food recipes for mixed dishes containing dry seasonings and rubs from McCormick Spices
(http://www.mccormick.com/Grill-Mates/Recipes). This is the lowest value, which would provide a worst-case scenario for
estimating exposure to a food additive in dry seasonings and rubs.

Oliphenol Page 1 6 of53 Hidrox®-GRAS

 As reported in GRN 726, the cumulative estimated daily intake of hydroxytyrosol from
existing dietary sources and the proposed uses described in GRN 726 (to deliver 5 to 10
mg/serving of hydroxytyrosol in 11 food categories) the highest 90th percentile per user
cumulative estimated dietary intake of hydroxytyrosol was determined as 55.1 mg/day among
teenagers ages 13 to 18 years (0.9 mg/kg-bw/day). The 90th percentile per user cumulative
estimated daily intake for the US population 2 years and older was determined as 52.4 mg/day
(0.9 mg/kg-bw/day). The resulting 90th percentile intake of Aqueous Olive Pulp Extract
(Hidrox®) will be approximately 1500 mg/person/day. For safety assessment purposes, the
highest intake for Aqueous Olive Pulp Extract of 1500 mg/person/day (25 mg/kg bw/day) is
considered.

3.3. Exposure from Presence in Food

The olive fruits and its oil have been used for centuries and are considered as an
important part of the Mediterranean diet. Given their organoleptic characteristics, olives require
processing prior to consumption (Brenes-Balbuena et al., 1992a; Brenes-Balbuena et al., 1992b;
Brenes-Balbuena et al., 1995; Goupy et al., 1 991; Robards et al., 1 999; Sciancalepore and
Langone, 1984). The phenolics in fruits or oil constitute a complex mixture. However, there are
some notable differences in composition between the two that are attributed to a series of
chemical or enzymatic alterations of some phenols during oil extraction. These changes include
hydrolysis of glycosides by glucosidases (Montedoro et al., 1 993; Angerosa et al., 1 996),
oxidation of phenolic compounds by polyphenol oxidases and, the polymerization of free
phenols (Ryan et al., 1999).
The quality of virgin oil is affected by the presence of phenolic compounds in olive fruits,
as these compounds are partly responsible for the stability and sensory characteristics. Given the
antioxidant properties, olive polyphenols, including hydroxytyrosol, have been the subject of
several clinical and preclinical investigations. Among the phenolic compounds present in raw
olive flesh, hydroxytyrosol and tyrosol are the most abundant and second most abundant
phenolics, predominantly occurring as esters. Hydroxytyrosol and tyrosol are structurally similar,
hydroxytyrosol possessing an extra hydroxy group in the meta-position. Both also occur as esters,
a notable example being the glycoside oleuropein. Oleuropein is an ester consisting of
hydroxytyrosol and elenolic acid.
Blekas et al. (2002) reported hydroxytyrosol (unbound) content of table olives as 250-750
mg/kg (approximately 0.5 mg/g) in two cultivars. Marsilio et al. (2001) reported that
consumption of 50 g black olive pericarp provides approximately 400 mg of phenolic substances,
while a similar quantity of extra virgin olive oil provides about 12 mg. As reported in GRN 726,
the levels of hydroxytyrosol in raw olives, including that present in conjugated forms, are of the
order of up to 1,800 mg/kg (DSM, 20 17). However, raw olives are not edible and need to
undergo extensive processing to produce the forms such as table olives and olive oil that are
most commonly consumed. Depending on the source and specific type of treatment,
hydroxytyrosol levels in table olives vary and can range from 400 to I 000 mg/kg. Processing is
also known to reduce hydroxytyrosol levels in olive oil and it ranges from 15 to 20 mg/kg.
In some Mediterranean countries, the average consumption of table olives is over 10
g/day, while individual consumption could be as high as 30 g/day. Similarly, in these countries,
consumption of olive oil on average is about 70 g/day and could be as high as 200 g/day for high
level consumers. Combining these occurrence levels and consumption data results in estimates of

Oliphenol Page 1 7 of53 Hidrox®-GRAS

average intakes of hydroxytyrosol in some Mediterranean countries of 12 mg/day, with the
potential for high level intakes to exceed 30 mg/day (DSM, 2017). For an individual weighing 60
kg, the combined hydroxytyrosol average and high intake in some Mediterranean countries can
range from 0.2 to 0.5 mg/kg bw/day.

Oliphenol Page 1 8 of53 Hidrox®-GRAS

Part IV- SELF LIMITING LEVELS OF USE
Oliphenol is unaware of any specific physical or technically impractical effects for Aqueous
Olive Pulp Extract (Hidrox®). Given the characteristic taste of Aqueous Olive Pulp Extract
(Hidrox®), it is unlikely that excessive amounts of this product are likely to be added to food
products.

Oliphenol Page 1 9 of53 Hidrox®-GRAS

Part V- EXPERIENCE BASED ON COMMON USE IN FOOD BEFORE 1958
The statutory basis for the conclusion of the GRAS status of Aqueous Olive Pulp Extract
(Hidrox®) in this document is not based on common use in food before 1958. The GRAS
assessment is based on scientific procedures. Notwithstanding this, the source material of the
extractt- olives- has been commonly present in food prior to 1958, as described below.

Oliphenol Page 20 of 53 Hidrox®-GRAS

Part VI- NARRATIVE
6.1. Traditional and Current Uses
The available information indicate that the olive tree was cultivated as early as 3500 BC
in Crete (Greek Islands). The tree has always represented a symbol of abundance, glory and
peace, and its leafy branches were used to crown the victorious in friendly games and war. It has
also been used to anoint the noblest of heads throughout history. During 1300 and 1200 BC,
Egyptian ruler, Ramses II, used olive oil for nearly every ailment. Shortly after the Iron Age
began ( 1100 - 750 BC), Greece became a large producer of olives/olive oil, spurred in the sixth
century BC by the prohibition of Solon (an Athenian lawmaker) of export of any agricultural
produce other than olive oil. Furthermore, throughout the Roman Empire, olive oil became a
popular staple in the diet. At present, approximately 90% of the world's olives are used in the
production of oil, with Spain, Italy, Greece and Portugal representing the main producers (Kiple
and Ornelas, 2000).
Olives cannot be eaten directly from the tree, as the fruit contain a bitter alkaloid. In
contrast to olives picked for oil production, olives selected for human consumption are picked
green and unripe. Because the raw green olives have a naturally bitter taste, the fruit cannot be
consumed unless further prepared/processed or cured. Edible olives preparation involves
pickling in a solution of lye to remove the bitter taste (rendered by oleuropein). This practice of
picking olives has been in use since Roman times. The traditional way of processing olives,
which is still a standard practice, involves three steps: blanching, salting and drying of mature
olives (Borzillo et al., 2000). The black color of table olives has nothing to do with ripeness, but
it is the result of exposure to air after lye extraction of olives. Black olives, used in the extraction
of oil, are allowed to ripen on the tree until after the time of the first frost.
This information suggests that the olive and its oil is consumed as a food without any
reported adverse effects.
6.2. Data Pertaining to Safety
In the following section, relevant toxicological and other studies on the olive and its
preparations are summarized in the order of their importance and in support of the conclusions
drawn in this assessment. This information is provided in the following sequence: published
pivotal studies, secondary published studies, corroborative unpublished studies and regulatory
agency assessments. Efforts have been made to present both the data supporting the safety as
well as any data on the adverse effects of olive fruit and its preparations.
6.2.1. Pivotal Studies of Aqueous Olive Pulp Extract
In a series of toxicity studies, conducted as per current accepted guidelines, the effects of
Aqueous Olive Pulp Extract (Hidrox®) has been investigated in animals and in vitro
experimental systems. All studies were conducted in strict compliance with Good Laboratory
Practices (GLPs), as defined by the FDA (FDA, GLP, Final rule, I 987). Toxicological
procedures in the acute studies reflect those described in the Redhook 2000. The overall findings
from all these studies with the subject of present GRAS, Hidrox®, are published in the journal
Drug and Chemical Toxicology by Christian et al. (2004). Additionally, these specific studies
and other available information on the olive and its preparations is extensively summarized in a
review article published in Food and Chemical Toxicology (Soni et al., 2006). A summary of the

Oliphenol Page 2 1 of53 Hidrox®-GRAS

published pivotal toxicity studies of Aqueous Olive Pulp Extract (Hidrox®) is provided in Table
8.

Table 8. Summary of Pivotal Toxicity Studies of Olive Pulp Extract (Hidrox®)

Duratio Animal
Study type Dose, Form and Route Summary Reference
n model
Hidrox® (0, 1 000, 1 500 or
Crl:CD
2000 mg/kg bw) per day No effects on body Christian
90 days Sprague-
Subchronic by oral gavage. (0, 60, 90 weight, body weight et al.,
Dawley
& 1 20 mg phenolics/kg gain, feed consumption 2004
rats
bw/day)
8/sex/
Hidrox® (0, I 000, 1 500 or
Reproductive group Estrous cycle of females,
2000 mg/kg bw) per day Christian
and Crl:CD mating & reproductive
by oral gavage. (0, 60, 90 1 4 days et al.,
Developmental Sprague- performance of males &
& 1 2 0 mg phenolics/kg 2004
Toxicity Dawley females not affected
bw/day)
rats
No adverse clinical
observations of
significant changes in
Developmental maternal body weights,
25 female
Hidrox® (0, 1 000, 1 500 or gravid uterine weights,
Toxicity Entire Crl:CD
2000 mg/kg bw) on day 6- or feed consumption. Christian
Designed in length of Sprague-
20 of gestation by oral No malformations et al.,
accordance gestation Dawley
gavage. Phenolic content occurred in pups. 2004
with FDA in rat rats per
of extract was 6%. Maternal & develop-
Redhook 2000 group
mental no observed
adverse effect level
(NOAEL) of extract >
2000 mg/kg/day
Crl:CD
Hidrox® (5000 mg/kg bw) Christian
Sprague- Tolerated after single
Short-term by oral gavage. (300 mg 29 days et al.,
Dawley and repeat dosing
phenolics/kg bw/day) 2004
rats
Single dose, Hidrox®
(aqueous olive pulp
extract) (500, I 000, or Christian
Acute No mortality, LDso of
Acute COi mice et al.,
2000 mg/kg bw) by oral study extract0> 2000 mg/kg
gavage. (0, 60, 90 & 1 20 2004
mg phenolics/kg bw dav)
Single dose, Hidrox® (0,
Crl:CD
I 000, 1 500 or 2000 mg/kg No adverse effects Christian
Acute Sprague-
Acute bw) by oral gavage. (0, 60, except soft or liquid et al.,
study Dawley
90 & 1 20 mg phenolics/kg feces 2004
rats
bw/day)
Mutae:eniciU and Genotoxicity Studies
Study type System Test sample Results Conclusion Reference
Each gram of For Salmonella, concentrations of
Salmonella test article I 000 µg/plate or more showed Positive at
and £. coli contains -24 mutagenic activity in the presence higher Christian
In vitro reverse mg of S9 activation. For £. coli, concentratio et al.,
mutation hydroxytyros twofold increase occurred in mean ns in certain 2004
assay ol (HT) & 60 number of revertants at strains.
mg phenolics. concentration of2500 µg/plate, in

Oliphenol Page 22 of 53 Hidrox®-GRAS

 the absence ofS9.
In each gram
Chromosome Extract showed significant increase
of test article, Extract was
aberration in in percentage of aberrant cells at
approximate! positive for Christian
Chinese 1 000 µg/mL in presence of S9 .
/11 vit,-o y 24 mg induction of et al. ,
Hamster Slight increases in numbers of
hydroxytyros chromosome 2004
Ovary polyploidy & /or endoreduplicated
ol (HT) & 60 aberrations.
(CHO) cells cells noted at I 000 µg/mL.
I mg nhenolics. I
No increase in micronucleated
polymorphic erythrocytes for any
In each gram
dose / time period in study.
of test article,
Hidrox® did not produce adverse
approximate! Christian
Micronucleus clinical or necropsy observations or
In vivo y 24 mg Negative et al.,
test in rats affect feed consumption values in
hydroxytyros 2004
any of animals. Body weight gains
ol (HT) & 60
for male & female rats receiving
mg phenolics.
5000 mg/kg bw reduced at third &
I fourth weeks of dosine. I
6.2.1.1 Subchronic Toxicity Study of Aqueous Olive Pulp Extract
In a subchronic study, Crl:CD® Sprague Dawley male and female rats (20/group/sex)
were administered (via gavage) a daily dose of Aqueous Olive Pulp Extract (Hidrox®)
(dissolved in aqueous 0.5% methy!cellulose) at levels of 0 (vehicle), 1000, 1500 and 2000 mg/kg
bw/day (0, 60, 90 and 120 mg/kg bw/day ofephenolics) for 90 days (Christian et al., 2004) (Table
8). The selection of the gavage route was based on the fact that ( I ) gavage administration most
simulates the method of intake in humans, consumed over a relatively short period of time; and
(2) high doses of the extract are not palatable. All standard parameters were studied. Physical
and ophthalmic examinations were conducted on all rats before and near the end of the study.
The following investigative parameters were recorded: daily clinical signs; weekly body weights
and feed consumption; hematology and serum chemistry determinations at the time of necropsy;
gross necropsy observations and histopathology of selected tissues at termination of the study.
Three satellite groups ( described later) were also attached to the main study.
Morbidity and mortality observations did not reveal any unusual findings. Excess
salivation and the presence of perioral substance noted in the treatment group were probably
associated with technical difficulties in administering the relatively thick granular suspensions of
the extract. All other clinical observations were considered unrelated to the test article.
Administration of the extract did not affect body weights, body weight gains or feed
consumption. A significant decrease in body weight gain noted for male rats in the l 000
mg/kg/day group on days 71-78 was considered unrelated to the test article because the value
was not dose-related. HIDROX® administration did not affect the organ weights.
Ophthalmologic observations did not reveal any treatment-related lesions (Christian et al., 2004).
Hematological investigations [WBC, RBC, hemoglobin, hematocrit, mean corpuscular
volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet counts,
prothrombin time and total serum protein] did not reveal any treatment-related differences
between the groups, except some incidental findings. Although not significant at two lower doses
( I 000 and 1500 mg/kg bw/day), a dose-related increasing trend in the number of RBC was noted
in female rats. The increase was significant in the 2000 mg/kg bw/day dose group. However, the

Oliphenol Page 23 of 53 Hidrox®-GRAS

values were within the range of historical control values. Increases in RBCs in female rats, in the
absence of changes in MCV, MCH, and MCHC were interpreted as a slight erythropoietic
stimulation of the bone marrow without any toxicological consequences. All other hematological
parameters in the male and female rats were unaffected (Christian et al., 2004).
Serum chemistry analysis for liver function tests such as aspartate aminotransferase
(AST), alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) showed a negative
trend. Reduction in these enzymes may be related to decreases in cholesterol levels. Levels of
ALT were significantly reduced in both male and female rats in all the extract treated groups. A
significant reduction in SDH levels was noted in female rats treated with 1500 and 2000 mg/kg
bw/day doses. Increases in plasma levels of ALT, AST and SDH are considered as markers of
hepatotoxicity. Decreases in these plasma enzymes (ALT, AST and SDH), observed in most of
the extract treated groups of male and female rats, were not considered to be a toxic
manifestation. The reasons for decreased activity could not be determined, although it may be
associated with the large quantities of olive polyphenols that have to be excreted in the bile. The
biliary excretion of large doses of the extract may also account for the slightly decreased serum
cholesterol levels in both male and female rats (significantly decreased in females at 2000 mg/kg
bw/day), because primary bile acids are synthesized by the liver from cholesterol (Tennant,
1 999). Although negative trends in plasma levels of ALT, AST and SDH were noted, all values
were within historical control value ranges. The changes were minor and do not appear to be a
toxicologically adverse response. Secondly, other biomarkers of liver function (serum bilirubin,
alkaline phosphatase, protein levels and histopathology) were unaffected by the extract. In
addition to the above-described changes, some incidental findings were noted. These incidental
findings were considered insignificant, as these changes were either not dose-related, of small
magnitude or not consistent between sexes (Christian et al., 2004).
Histopathological examinations revealed minimal or mild focal hyperplasia of the
mucosa! squamous epithelium of the limiting ridge of the forestomach of male and female rats in
the high dose (2000 mg/kg bw/day) group. Similar changes with a very low incidence of
hyperplasia were noted in animals treated with mid dose (1500 mg/kg bw/day) of the extract and
in the control group. All other microscopic changes that were noted in the various organs and
tissues examined were considered spontaneous in origin, incidental to treatment, and not
associated with any systemic toxicity of the extract. Although focal, minimal or mild hyperplasia
of the mucosa! squamous epithelium of the limiting ridge of the forestomach was noted, this type
of change is often associated with irritation of this area of the gastric mucosa and was considered
to be due to local irritation by the large intubated volume of viscous, granular formulation. Based
on the findings from this study, the no observed adverse effect level (NOAEL) is established as
2000 mg/kg bw/day, the highest dose tested (Christian et al., 2004). The results of this study
suggest that the resulting all user maximum intake of 25 mg/kg bw/day from the proposed uses
of Aqueous Olive Pulp Extract (Hidrox®), the NOAEL is 80-fold lower.
6.2.1.2 Developmental and Reproductive Toxicity of Aqueous Olive Pulp Extract
In a study designed to investigate potential reproductive and developmental toxicities,
Aqueous Olive Pulp Extract (Hidrox®) was administered by oral gavage to Crl:CD® Sprague
Dawley rats (Christian et al., 2004) (Table 8). In this study, rats ( 8/sex/group) were assigned to
five dose groups received the extract at dose levels of 0, 500, 1000, 1500 and 2000 mg/kg
bw/day once daily for I 4 days before cohabitation and continued until the day before necropsy
(males were euthanized after being administered a total of 49 daily doses of the extract; females

Oliphenol Page 24 of 53 Hidrox®-GRAS

were euthanized after completion of the 22-day post-partum period). Clinical observations were
recorded daily, while body weights of the male and female rats were recorded on days 1 through
7 and 14, and then weekly for the males and daily for the females during gestation and on days 0,
4, 7, 10 and 14 of lactation. Female rats were also observed for estrous cycling (two weeks each
pre-treatment and treatment), adverse clinical signs during parturition, maternal behavior,
duration of gestation, litter sizes and pup viability at birth and maternal behavior. Pups in each
litter were counted and clinical observations were recorded once daily during the one-week
postpartum period (birthe= day zero postpartum).
In this study, on day 21 postpartum, all F 1 generation pups were weaned. Two male and
two female F 1 generation pups per litter (i.e., a total of 160 rats, 80 rats per sex) were selected
for a week of daily gavage treatment and recording of clinical signs, body weights and viability
before being euthanized and necropsied on post-partum day 28. All other pups were subjected to
gross necropsy on post-partum day 21. On day 28 postpartum, the retained pups were euthanized
and examined for gross lesions at necropsy. Male rats in the main study were killed after
completion of the cohabitation period. Surviving female rats in the main study were killed after
completion of the 22-day postpartum period. All main study rats were subjected to a gross
necropsy of the thoracic, abdominal and pelvic viscera (Christian et al., 2004).
In the Fo generation, all male rats survived to the scheduled euthanasia. Occasional
instances of excess salivation and non-dose-related increases in body weight gains were the only
findings associated with the Aqueous Olive Pulp Extract administration. Absolute and relative
feed consumption values for the entire dose period were not affected. Mating and fertility
parameters for the male rats were comparable among the five dose groups. All necropsy
observations were considered unrelated to the test article, as also were the terminal body weights,
and the weights of the paired epididymides and testes. The ratios of the male reproductive organ
weights to the terminal body weights were comparable among the five dose groups (Christian et
al., 2004).
Except for one female, all female rats in the Fo generation survived until the scheduled
euthanasia. The one dam in the 1000 mg/kg bw/day group was found dead on the first day of
lactation. The death was attributed to a torsion of the right uterine horn, a finding unrelated to the
test article. Incidental observations of excess salivation were noted in the female rats in the 1000,
1500 and 2000 mg/kg bw/day dose groups during the gestation period and in one or two female
rats in all extract treated groups during the precohabitation and lactation periods. A tendency for
increased body weight gains for the entire precohabitation period was also noted for these groups.
Body weight gains for the entire gestation period were comparable among the groups, although
the body weight gains for the entire lactation period were reduced in the 1500 and 2000 mg/kg
bw/day dose groups, as compared with the control. Absolute and relative feed consumption
values for the precohabitation, gestation and lactation periods for the female rats were
comparable among the five dose groups, again indicating that the increased weight gains were
probably associated with the nutritional value of the extract or increased water retention. Estrous
cycling, mating and reproductive performance of the female rats were not affected by the extract
treatment. Small reductions (<10%) in pup body weights on lactation days 7, 14 and 21 in the
1000, 1500 and 2000 mg/kg bw/day dose groups were noted. All other delivery and litter
observations were comparable among the five dose groups (Christian et al., 2004).
Based on the findings from this study, Aqueous Olive Pulp Extract does not appear to be
a reproductive or developmental toxicant. As no toxic effects were noted in the parental rats

Oliphenol Page 25 of 53 Hidrox®-GRAS

during the first two weeks of treatment, similar doses ( 0, I 000, 1500 and 2000 mg/kg bw/day) of
Aqueous Olive Pulp Extract ( Hidrox®) were recommended for use in the developmental toxicity
( see below) study and 90-day toxicity ( described above in section 6.2.1.1.) study in rats. The
high dose of 2000 mg/kg bw/day is generally considered the highest dose necessary for studies
of this type ( Christian et al., 2004).
In a follow-up to the above described study, Christian et al. ( 2004) evaluated the
developmental toxicity ( embryo-fetal toxicity and teratogenic potential) of Aqueous Olive Pulp
Extract ( Hidrox®) in Crl:CD® Sprague-Dawley rats ( Table 8). This study was designed as per
FDA Redhook 2000 recommendations ( FDA, 2004). For these investigations, time-mated female
rats ( 25/group) were randomly divided into four groups. On days 6 through 20 of presumed
gestation, the extract or the vehicle ( 0.5% w/v methylcellulose) was administered via gavage
once daily at dose levels of 0, 1000, 1500 and 2000 mg/kg bw/day. The phenolic content of the
extract was 6% ( 60 mg/g).
One dam in the high dose ( 2000 mg/kg bw/day) group was euthanized, on day 21 of
gestation, because of premature delivery of litter before scheduled Caesarean-sectioning. No
abnormal findings were noted for this dam or its litter. All other rats survived until scheduled
Caesarean sectioning. No adverse clinical or necropsy observations or significant differences in
maternal body weights, body weight gains, gravid uterine weights, corrected maternal body
weights or body weight gains or absolute or relative feed consumption values were noted
between the groups. Caesarean-sectioning observations were based on 23, 22, 22 and 24
pregnant rats with one or more live fetuses in the four respective groups. The extract treatment
did not affect litter parameters at any of the doses. No treatment-related increases in gross
external, soft tissue and skeletal fetal alterations ( malformations or variations) were observed. A
significantly increased mean number of corpora lutea of the 2000 mg/kg be/day dose group was
well within the historical range of 14.5-20.1 per litter of the laboratory and was attributed to two
females that had 27 or 30 corpora lutea. Based on the findings from this study, the maternal and
developmental NOAEL of Aqueous Olive Pulp Extract ( Hidrox®) was 2000 mg/kg bw/day, the
highest dose tested ( Christian et al., 2004).
6.2.1.3 Genotoxicity Studies of Aqueous Olive Pulp Extract
In order to investigate genotoxic potentials, Aqueous Olive Pulp Extract ( Hidrox®) was
subjected to three mutagenicity assays: a bacterial reverse mutation assay, a chromosome
aberration assay and a rat micronucleus assay ( Christian et al., 2004; Soni et al., 2006) ( Table 8).
6.2.1.3.1 Bacterial Reverse Mutation Assay (Ames test)
In an in vitro study, Aqueous Olive Pulp Extract ( Hidrox®) was tested for potential
mutagenicity in a bacterial reverse mutation assay employing Salmonella typhimurium strains
TA97, TA98, TAI 00 and TA1535 and Escherichia coli strain WP2 uvrA ( 328), in the presence
and absence of S9 ( Table 8). The test was performed at different concentrations of the extract ( 0,
5, I 0, 50, I 00, 500, I 000, 2500 and 5000 µg/plate) and concentrations of 50, I 00, 500, 1000
and 2500 µg/plate were used in the more sensitive confirmatory preincubation assay. A result
was classified as positive (i.e., mutagenic) if the average number of revertants in any strain at
any test article concentration was at least two times greater than the average number of
revertants in the concurrent vehicle control and/or there was a concentration-related increase in
the mean revertants/plate in that same strain. In the Ames test, at concentrations of I 00 µg/plate
or above of the extract, precipitates were observed. As determined by a concentration-related

Oliphenol Page 26 of 53 Hidrox®-GRAS

reduction in the mean number of revertants/plate and/or the reduction of the microcolony
background lawns, toxicity was noted at concentrations of 500 µg/plate or above. Evidence of
mutagenic activity was only detected in strains T A98 and TA 100 at doses of 1000 and 2500
µg/plate (in the presence of S9 activation for both the strains). The number of revertants per
plate at concentrations of 0, 1000 and 2500 µg/plate for TA 98 were reported as 23, 52 and 133,
respectively. Similarly, the number oferevertants per plate for the strain TAI00 was reported as
1 57,e372 and 1051, respectively (Christian et al., 2004).
In experiments with E. cotli, no mutagenicity was noted at any of the concentrations
tested, except for a two-fold increase in mean number of revertants at concentration of 2500
µg/plate, in the absence of S9. The positive results were confirmed in the more sensitive
preincubation test, but only with metabolic activation. Some inconsistencies between the
regular and repeat trials were noticed. The investigators stated that the antibacterial properties
of the test article, and observation of positive findings only at one or two concentrations, where
precipitates and toxicity occurred, tended to complicate the interpretation of these mutagenic
findings. The investigators concluded that under the conditions of the study, equivocal evidence
of mutagenic activity of Aqueous Olive Pulp Extract (Hidrox®) was detected in S. typhimurium
strains TA98 and TA! 00 (Christian et al., 2004).
6.2.1.3.2 Chromosomal Aberrations Assay
In another in vitro genotoxicity assay, the effects of Aqueous Olive Pulp Extract
(Hidrox®) on chromosome aberrations in Chinese Hamster Ovary (CHO) cells were
investigated, in the presence and absence of metabolic activation system (S9) (Christian et al.,
2004; Soni et al., 2006) (Table 8). The cell cultures were treated with 0, 1 0, 50, 100, 300, 600
and 1000 µg of the extract/ml; positive and negative (vehicle, dimethyl sulfoxide) controls were
also included. Cultures were incubated with the extract for approximately three hours, after
which the treatment medium was washed and replaced with a fresh culture medium. Cells were
sampled at a time approximately 20 hours from the beginning of treatment. Approximately two
hours prior to harvest, Colcemid® was added to arrest cells in metaphase. Test article
concentrations of 100, 300 and 1000 µg/ml were assessed for effects on mitotic index, polypoid
cells and aberrations (chromatid and chromosome breaks/exchanges).
In this study, no clear evidence of test article-related toxicity, as evidenced by the
confluence rate or mitotic index, was observed at any concentration level of the extract. The
extract elicited a significant increase in the percentage of aberrant cells at I 000 µg/ml in the
presence of S9. At this concentration, slight increases in the numbers of polyploid and/or
endoreduplicated cells (numerical chromosome changes) were also noted. The positive
response was associated with the presence of test article precipitate during treatment. Based on
the findings of this study, the investigators concluded that Aqueous Olive Pulp Extract
(Hidrox®) was positive for the induction of chromosome aberrations in CHO cells (Christian et
al., 2004).
6.2.1.3.3 Micronucleus Assay
As described above, both in vitro assays (bacterial reverse mutation and CHO
chromosome aberration) exhibited evidence of mutagenic activity of Aqueous Olive Pulp
Extract (Hidrox®) (Table 8). However, the positive results in both these assays were only
observed at one or two of the highest concentrations of Aqueous Olive Pulp Extract (Hidrox®),
where slight amounts of precipitation were noted, and were confirmed only in the presence of

Oliphenol Page 27 of 53 Hidrox®-GRAS

S 9 metabolic activation. The effect of the precipitate on the outcome of the results could not be
determined. Given the confounding factors present in the bacterial and tissue culture assays, a
study in live animals, i.e., a micronucleus assay in rats for Aqueous Olive Pulp Extract
(Hidrox®) was undertaken (Christian et al., 2004).
In the micronucleus assay, adult male and female Crl:CD® Sprague Dawley IGS BR
rats were used. Single and 2 8 consecutive daily doses of 1000, 1500 and 2000 mg/kg bw/day,
and 2 9 consecutive days of 5000 mg/kg bw/day, of Aqueous Olive Pulp Extract (Hidrox®)
were gavaged. The number of rats included in each group were as follows: vehicle or negative
control ( J O/sex); 1000 mg/kg bw/day dose (5/sex); 1500 mg/kg bw/day dose (5/sex); 2000
mg/kg bw/day dose (14/sex); 5000 mg/kg bw/day dose (5/sex; additional group of rats tested
after 2 9 consecutive days of treatment); and positive control dose (5/sex- treated with
cyclophosphamide). In addition to euthanization at 24 hours from all groups, rats in negative
control dose (5/sex) and receiving 2000 mg/kg bw/day (7/sex) were also euthanized at 4 8 hours.
Following euthanization of the rat at approximately 24 hours after the last dose, bone marrow
samples from the femur of each rat were collected for further analysis (Christian et al., 2004).
A minimum of 2000 polychromatic erythrocytes (PCEs) was scored for micronuclei.
The number of PCEs among 500 total erythrocytes (expressed as PCEs fraction) was
determined for each animal. The number of micronucleated normochromatic erythrocytes
(NCEs) was also recorded. The extract did not produce adverse clinical or necropsy
observations or affect absolute or relative feed consumption values. Body weight gains for the
male and female rats in the highest dose group were reduced at the third and fourth weeks of
daily dose, as compared with previous weeks. The numbers of micronucleated PCEs were not
significantly increased in any of the extract treated groups, as compared to the control. The ratio
of PCEs to NCEs was not affected by the administration of the extract. The positive control, on
the other hand, elicited clear increases in micronucleated-PCEs in both sexes, without causing
excessive toxicity. The micronucleus assays after repeated dosages of the extract confirmed the
results achieved after single dosages, and it was concluded that the extract was not mutagenic.
The results of this study demonstrate that Aqueous Olive Pulp Extract (Hidrox®) was negative
in the micronucleus assay at 24 and 4 8 hours after a single dose of 1000, 1500 or 2000 mg/kg
and also at 24 hours after 2 8 daily doses of 0, 1000, 1500, 2000 or 5000 mg/kg (Christian et al.,
2004).
6.2.1.4 Acute and Short-term Toxicity Studies of Aqueous Olive Pulp Extract
In an acute toxicity study in CRL:CD I ICR (BR) mice, oral gavage administration or
dermal application of a single dose of Aqueous Olive Pulp Extract (Hidrox®) at levels of 500,
1000 or 2000 mg/kg failed to produce any adverse effects on clinical observations, body weight,
body weight changes or gross pathology (Table 8). No mortality was noted in any of the
treatment groups, suggesting that the LDso of the extract is greater than 2000 mg/kg in mice.
The extract was well tolerated in mice (Christian et al., 2004). In another study, oral
administration of a single gavage dose of Aqueous Olive Pulp Extract (Hidrox®) (Christian et
al., 2004) at levels of 0, 1000, 1500 or 2000 mg/kg to Crl:CD® Sprague Dawley rats
(5/sex/group) did not cause any adverse effects except soft or liquid feces (Christian et al.,
2004).
In a short term study that was conducted as part of a micronucleus assay, Crl:CD®
Sprague Dawley rats (5/sex) were administered (gavage) a single dose of 5000 mg olive pulp

Oliphenol Page 28 of 53 Hidrox®-GRAS

extract/kg, and the rats were observed for six days, after which the 5000 mg/kg dose was given
for 29 consecutive days (Christian et al., 2004) (Table 8). No mortality or clinical signs of
toxicity were noted. Both male and female rats continued to gain weight, although at a reduced
rate as compared to the control rats. Absolute and relative feed intake were similar to that in the
control animals. The findings from this study show that the LDso of Aqueous Olive Pulp Extract
(Hidrox®) is greater than 5000 mg/kg and suggests that Hidrox® is practically nontoxic
(Christian et al., 2004).
6.2.1.5 Absorption Study of Aqueous Olive Pulp Extract
As part of the above described subchronic toxicity study, Christian et al. (2004) also
investigated the absorption of hydroxytyrosol from exposure to Aqueous Olive Pulp Extract
(Hidrox®). For this assessment, Sprague Dawley rats (6/sex/group) were administered Aqueous
Olive Pulp Extract (Hidrox®) at dose levels of 1 000, I 500 and 2000 mg/kg bw/day
(corresponding to hydroxytyrosol at 24, 36 and 48 mg/kg/day, respectively) by oral gavage for
90 days (Christian et al., 2004). Blood samples were collected on Day 90, prior to dosing and at
0.5, I , 2, 4 and 8 hours post-dose. Pre-dose plasma samples contained no measurable mean
concentrations of hydroxytyrosol, suggesting minimal carry-over of hydroxytyrosol from prior
doses. The findings from this study suggest that hydroxytyrosol is rapidly absorbed, and mean
concentrations were measurable through I to 4 hours at dose levels of I 000 and 1500 mg/kg
bw/day and through 8 hours at 2000 mg/kg bw/day for 90 consecutive days. This study also
indicate that hydroxytyrosol is unlikely to accumulate in the body.
6.2.1.6 Human Study of Aqueous Olive Pulp Extract
In a double-blind, randomized, placebo-controlled trial, Bitler et al. (2007) investigated
the effects of a polyphenolic-rich olive extract (freeze-dried olive vegetation water; Hidrox) on
a series of parameters in male and female subjects (n= l 05; age 55-75 years) with osteoarthritis
and rheumatoid arthritis. The olive water fraction was reported to contain at least 6% simple
phenols and polyphenols. The subjects in the treatment group (n=5 I ) received 400 mg of the
freeze-dried extract/day for eight weeks. Of the 105 subjects, 47 in the placebo group and 43 in
the treatment group completed the study. Serum samples were analyzed for clinical and
biochemical parameters. The rheumatoid arthritis subjects in the extract treatment group
showed significant decreases in serum homocysteine levels after eight weeks of treatment.
No significant changes in any other clinical marker, including markers of renal (serum
blood urea nitrogen and creatinine) and hepatic function (aspartate aminotransferase, alanine
aminotransferase, alkaline phosphatase, and total bilirubin) were noted at any time during the
study (Bitler et al., 2007). These observations support safety of the supplement. Overall, the
participants tolerated the placebo and supplement well; only two participants, one from each
group (placebo and supplement), complained of heartburn at the two-week visit. This problem
was alleviated when the participants took the placebo or supplement with food. The results of
this study did not reveal any adverse effects of the olive extract in the arthritis subjects.
Although the levels of hydroxytyrosol were not reported in the publication, given the affiliation
of the authors of this study, the extract used in this study appears to be the subject of this
present GRAS assessment and the resulting intake of hydroxytyrosol appear to be
approximately IO mg/person/day.
6.2.2. Secondary Pivotal Published Studies of Olive Extracts

Oliphenol Page 29 of 53 Hidrox®-GRAS

 In addition to the specific studies on Aqueous Olive Pulp Extract (Hidrox®), extensive
safety analyses have also been conducted on similar substances derived from olive fruit,
including other products such as elaYida™ 40% ( the subject URN 726), a polyphenol
preparation made from olive fruits using solvent-free process; elaVida™ 15% ( contains 15%
hydroxytyrosol). Moreover, published studies with chemically pure hydroxytyrosol ( subject of
GRN 876 and GRN 600) have also been considered in establishing the safety of Aqueous Olive
Pulp Extract (Hidrox®). While these substances are not identical in composition, findings from
all these studies contribute to the total weight of safety evidence for the present GRAS
assessment. The current safety assessment is focused on the safety evaluation of Aqueous Olive
Pulp Extract (Hidrox®) and the main phenolic component hydroxytyrosol and the supportive
data contained in this dossier.
6.2.2.1. Subchronic Toxicity Studies
In a 13-week rat study, Heilman et al. ( 2015) investigated the safety of olive extract H35
containing 35% hydroxytyrosol ( Table 9). In this study, olive extract H35 was administered
orally ( gavage) to Wistar rats for 13 weeks, followed by a 4-week treatment-free period, at doses
of 0, 345,t691 and 138 1 mg/kg bw/day. The doses were equivalent to hydroxytyrosol content of
0, 125, 250 and 500 mg/kg bw/day. The study was performed following OECD guideline 408
and GLP with inclusion of additional elements. These included neurobehavioral observations,
seminology, estrous cycling and a MNT genotoxicity element. Also, blood samples were
collected 30 minutes after dosing one day in weeks 4, 8, and 13 for hydroxytyrosol analysis.
No mortality or morbidity was observed during the study period (Heilman et al., 2015). At
termination, reductions in body weight of 9%, and a statistically significant reduction in body
weight gain of approximately 17% ( p< 0.05) at week 13 were noted in high dose males ( 500 mg
hydroxytyrosol/kg bw/day). In addition to this, a statistically significant increase in relative
weights of the liver, heart, and kidneys of high dose males and females were noted. These
changes were not accompanied by pathological or clinical observations and a trend towards
reversal was observed in the recovery phase. The results of this study show that olive extract
H35 was well-tolerated and no toxicologically significant treatment-related changes were
observed in condition and appearance of rats, neurobehavioral outcomes, motor activity
assessments, functional observational battery ( FOB), food intake, ophthalmoscopic examinations,
hematology, clinical chemistry, urinalysis, necropsy findings. Additional parameters studied
such as reproductive effects ( including estrous cycle assessment and sperm analysis) did not
reveal observation of any statistically significant changes between treated animals and control
(Heilman et al., 2015).
Based on statistically significant reductions in body weight gain and decreased absolute
body weight in males, the investigators determined the lowest observed adverse effect level
( LOAEL) of olive extract H35 as 1381 mg /kg bw/day ( 500 mg hydroxytyrosol/kg bw/day). The
investigators reported that, based solely on the reduction in body weight and body weight gain in
the high dose males, it is conservatively concluded that the NOAEL of olive extractH35 is 691
mg/kg bw/day ( 250 mg hydroxytyrosol/kg bw/day) (Heilman et al., 2015).
Aunon-Calles et al. ( 2013a) conducted a subchronic toxicity study with pure hydroxytyrosol
in accordance with OECD Guideline 408, and GLP ( Table 9). In this study, hydroxytyrosol was
administered orally daily by gavage to groups of Wistar rats ( I 0/sex/group) at dose levels of 0, 5,
50, and 500 mg/kg bw/day for 90 consecutive days. Additional animals ( 5 rats/sex) in groups I

Oliphenol Page 30 of 53 Hidrox®-GRAS

and 4 were included for a four-week recovery period. During the course of the study, no
mortality was noted in any group. No relevant treatment-related clinical signs were recorded.
Salivation was recorded in all animals treated at the high dose and sporadically in some animals
from groups treated at the intermediate- and low-doses. This phenomenon was attributed to the
bitter taste of hydroxytyrosol and/or the physical characteristics of the formulation ( slightly oily
and dense). In the high dose treated group ( 500 mg/kg bw/day), slightly but significant, lower
body weight ( 14%) in males and body weight gain in males and females were observed. The
decrease in male body weight is supported by the Heilman et al. ( 20 15) study where a similar
decrease ( 17%) in male rat body weight following administration of hydroxytyrosol at dose
levels of 500 mg/kg bw/day was noted.

Table 9 Summarv of secondary p·1vota I Pub I'1shed S ubc h 1·omc T0XICit

. . . y Stud'1es
Test substance; Study type; Animals (sex/group);
Results NOAEL Reference
Route; Duration Doses (me/k2 mw/day)
I 0/sex/group, plus recovery Based on results excluding
Olive extract H35; Rat sub-
animals; 0, 345, 691 and MNT phase: 691 mg/kg bw/day
chronic; Gavage; 90 days Heilman et al.,
1 3 8 1 olive extract H35; 0, (250 mg hydroxytyrosol) (slight
plus 28-day treatment-free 20e1 5
1 25, 250 and 500 as reduction in male weight at 500
period
hydroxytyrosol mg/kg bw/day)
I 0/sex/group, plus recovery 500 mg/kg bw/day (minor
Hydroxytyrosol; Rat sub- Aunon-Calles
animals; 0, 5, 50, and 500 changes observed were
chronic; Gavage; 90 days et al., 20 I 3a
pure hydroxytyrosol considered not adverse)

The hematological and clinical chemistry changes revealed higher MCY and MCH in
females treated at the high- and intermediate-doses; higher HFR and WBC values in females
treated at the high dose; lower creatinine and higher albumin values in males treated at the high
dose; and higher calcium values in males treated at intermediate- and high-doses ( Aunon-Calles
et al., 2013a). As these significant changes in hematology and clinical chemistry parameters were
not observed in both sexes, were of small magnitude, lacked correlating changes in other clinical
parameters and were not noted in a dose-related manner, or were not associated with microscopic
changes in the related organs, they were considered as incidental changes/biological variations
and not treatment-related adverse effects ( Aunon-Calles et al., 2013a). Among the organ weights,
higher kidney weights were observed in males and females from the 500 mg/kg group.However,
no alterations in this organ were observed on histopathological examination and this finding was
not considered to be toxicologically relevant. Microscopic observations did not reveal any
morphological alteration in any of the organs or tissues examined. Based on the results obtained,
daily oral administration of hydroxytyrosol to rats for a period of 13 weeks did not induce effects
that can be considered of toxicological relevance. Hence, the investigators proposed the dose of
500 mg/kg bw/day as the NOAEL.
In a recent subchronic toxicity study conducted as per OECD-40 8 guidelines, Rodriguez­
Lara et al. ( 20 1 9) investigated the effects of an aqueous virgin olive oil ( YOO) extract rich in
hydroxytyrosol in rats. The extract contained an initial concentration of 15% of hydroxytyrosol.
For this study, 80 Wistar SHD rats were divided into four group ( 10/sex/group) and were
administered YOO at levels of 0, 100, 300 and 1000 mg/kg bw/day in the drinking water. The
YOO concentration in drinking water was adjusted such that the daily exposure was achieved.
All standard parameters, as per OECD guidance, during the course of this study and at
termination were measured. No toxic effect of YOO extract rich in hydroxytyrosol were noted
after the sub-chronic supplementation during 90 days with 100, 300 and 1000 mg/kg bw/day,

Oliphenol Page 3 1 of 53 Hidrox®-GRAS

according to the OECD-408 guidelines. Although minor hematological and biochemical
differences were observed between the control and the YOO extract-supplemented groups, these
changes did not follow a dose-dependent pattern, and all evaluated clinical, hematological,
biochemical, and histological parameters were within normal ranges in all of the animals, which
indicates no toxicological effect of YOO extract at the length and dosages examined. The
investigators concluded YOO extract containing 15% of hydroxytyrosol did not induce effects
that can be considered of toxicological relevance, and proposed a NOAEL dose as I 000 mg/kg
bw/day of pure hydroxytyrosol.
6.2.2.2. Mutagenicity and Genotoxicity Studies
In an extensive article, Kirkland et al. (2015) reviewed the available studies and also
published mutagenicity and genotoxicity related findings from studies conducted by their group.
These investigators noted that pure hydroxytyrosol, and an olive extract containing 1 5%
hydroxytyrosol, both induced micronuclei in cultured cells in vitro, but show that these responses
were either due to high levels of cytotoxicity or to reaction of hydroxytyrosol with culture
medium components to produce hydrogen peroxide. However, both extracts (15% and 40%)
were negative in robust Ames tests. Another olive extract containing 40% hydroxytyrosol also
induced micronuclei in vitro, probably via the same mechanism. Kirkland et al. (20 1 5) concluded
that the amounts of hydrogen peroxide produced at the concentrations tested may well be
sufficient to account for the increased micronucleus frequencies seen with olive extracts in the
absence of S 9.
A summary of in vivo genotoxicity studies summarized in Kirkland et al. (2015) are
provided in Table 10. This table also includes pivotal genotoxicity studies ofHidrox® that are
described earlier. In the in vivo study by Kirkland et al. (2015), an olive extract containing 15%
hydroxytyrosol did not induce micronuclei in rat bone marrow after four weeks of dosing at
levels up to 561 mg hydroxytyrosol/kg bw/day. In a 90-day repeat-dose study, olive extract
containing 35% hydroxytyrosol produced increased rat bone marrow micronucleus frequencies at
250 and 500 mg hydroxytyrosol/kg bw/day, but the results were questionable for various reasons.
However, when two different batches of this extract were tested in acute micronucleus studies at
doses up to 2000 mg hydroxytyrosol/kg bw, giving plasma exposures that exceeded those in the
90-day study, negative results were obtained. Based on the weight of evidence, these
investigators concluded that the olive extracts tested are not genotoxic at high doses in vivo, and
any genotoxic risks for human consumers are negligible (Kirkland et al., 2015).
As described in GRN 726 (DSM, 2017), the genotoxicity studies of olive extract provide
a conclusion of a lack of genotoxic concern on a Weight of Evidence basis. It was also stated in
GRN 726 that, based on the overall genotoxicity evaluation, the notifier concluded that for olive
extracts in general, the specific olive extract from the process used to make olive extract H40,
and for the main olive polyphenol (hydroxytyrosol), that any genotoxic risks for human
consumers are negligible. Negligible risk is usually regarded as the lowest level of risk.
Subsequent to the Kirkland et al. (2015) publication that went to press in 2014, an in vivo
chromosome aberration test in rats (Dolan et al., 2014) was published. At the oral limit dose of
2000 mg/kg bw, hydroxytyrosol also did not induce chromosome aberrations in bone marrow
cells.
In the study by Dolan et al. (2014), potential clastogenic effects of pure hydroxytyrosol in
a bone marrow chromosome aberration study in rats was investigated. The study was conducted

Oliphenol Page 32 of 53 Hidrox®-GRAS

as per OECD Guideline 475 (mammalian bone marrow chromosome aberration test) in rats with
the oral limit dose of 2000 mg/kg bw. Hydroxytyrosol, dissolved in distilled water, was
administered via gavage to two groups of rats (5/group/sex). Two groups of five animals per sex
(negative controls) were dosed with vehicle (distilled water) only. Five male and five female rats
served as positive controls and received 40 mg/kg bw cyclophosphamide in saline by
intraperitoneal injection. Four hours before scheduled euthanization (24 and 48 hour time points
for both treated and negative control animals and 24 hours for the positive control group), the
rats received 2 mg/kg colchicine (a metaphase arresting agent) by intraperitoneal injection. At
tennination, femurs were removed and bone marrow was harvested. The oral limit dose of 2000
mg hydroxytyrosol/kg bw was well tolerated by most rats. However, some rats exhibited clinical
signs that abated within 24 hours. Treatment with hydroxytyrosol did not significantly enhance
the number of aberrant cells or the mitotic index 24 or 48 hours post-dose. The positive control
(cyclophosphamide) induced the expected increase in chromosomal aberrations and a decrease in
the mitotic index, confirming the validity of the assay. The investigators concluded that an oral
limit dose of 2000 mg hydroxytyrosol/kg does not induce chromosome aberrations in bone
marrow cells of the rat. This suggest that hydroxytyrosol is not a clastogen in vivo.

Table to. s ummary of Ill vivo Genotoxicitv Studies Reviewed bv Kirkland et al. (2015)
Results
Animals (sex/group);
Reference Study type; Route; Duration NOAEL0for
Doses (mg/kg bw/day)
hydroxvtvrosol
Studies with Olive Extracts, includin!! H35, H40 and H4 0 Mild Process Conditions and Hidrox
MNT phase: 1 25
I 0/sex/group, plus recovery
Kirkland et MNT element in rat sub-chronic; Gavage mg/kg bw/day
animals;
al. (20 1 5 ) H 3 5 ; 9 0 days, 24 hours following last dose Positive MN effect
0, 1 25, 250, and 500
at higher dosages
Kirkland et Classic acute MNT in rat; Gavage H40; 7 males/group;
Non-genotoxic
al., 20 1 5 Single dose 24 and 48 hours post dose 0, 500, I 000, and 2000
Kirkland et Classic acute MNT in rat; Gavage H40
7 males/group;
al. (2015) MPC; Single dose 24 and 48 hours post Non-genotoxic
0, 500, 1 000, and02000
dose
Up to 2000 mg/kg bw in
Christian et Acute MNT in rat; Gavage Hidrox; Single Non-genotoxic at up
terms of extract HT content
al. (2004) dose 24 and 48 hours post dose to 48 mg/kg bw
2.4%
Up to 5000 mg/kg bw/day in
Christian et Rat sub-acute; Gavage Hidrox; 4 weeks, Non-genotoxic at up
terms of extract
al. (2004) 24 hours after last dose to 1 20 mg/kg bw
hvdroxvtvrosol content 2.4%
Studies with Hydroxytyrosol
I 0/sex/group, plus recovery
Kirkland et Rat sub-acute; Gavage Hydroxytyrosol Non-genotoxic at
animals
al. (20 1 5 ) 1 5% SD; 4 weeks, 2 7 days ?:561 mg/kg bw/day
0, 62, 187, and 561
Rat bone marrow chromosome aberration;
Dolan et al. 2000 mg/kg bw of
Gavage Hydroxytyrosol; Single dose 24 Non-clastogenic
(2014) hydroxytyrosol
and 48 hours
MNT=m1cronucleus test

Aunon-Calles et al. (201 3b) investigated the genotoxic and mutagenic potential of
hydroxytyrosol, using well-established in vitro models, i.e., the chromosomal aberration assay
and the Ames test (by using the S. typhimurium TA 1 00, TA98, TA1535, and TA 1537 strains
and E. coli WP2(pKM 1 0 1 )), with and without S9-induced metabolic activation). No dose
response for hydroxytyrosol was observed in any of the tested bacterial strains. The investigators

Oliphenol Page 33 of 53 Hidrox®-GRAS

noted that, even though it cannot be ruled out, prolonged exposure to hydroxytyrosol and its
metabolites might have untoward effects. However, the results of this study indicate that
hydroxytyrosol is non-genotoxic and non-mutagenic at concentrations that far exceed those
attainable after intake.
In another in vitro study, Aunon Calles et al. (2013b) also investigated the potential of
hydroxytyrosol to induce chromosomal aberrations in human lymphocytes in the absence and
presence of metabolic activation by S9 mix. The highest treatment concentration in this study,
1542 µg/mL ( -10 mM) was chosen based on the molecular weight of the test item and with
respect to the OECD Guideline for in vitro mammalian cytogenetictests. No visible precipitation
of the test item in the culture medium was observed. No relevant influence on osmolality or pH
value was observed. In the absence of S9 mix one statistically significant increase in the number
of aberrant cells, excluding gaps (9%) was observed after treatment with 503.5 µg/mL. In the
presence of S9 mix after treatment with 287. 7 and 503.5 µg/mL two statistically significant
increases (3.5% and 4.5% aberrant cells, excluding gaps, respectively) were observed. These
values exceeded the range of the laboratory historical solvent control data (0.0-3.0% aberrant
cells, excluding gaps). No evidence of an increase in polyploid metaphases was noticed after
treatment with the test item as compared to the control cultures. The positive controls showed
distinct increases in cells with structural chromosome aberrations.
In summary, the available evidence from mutagenicity and genotoxicity studies revealed
that in in vitro Ames assays olive extracts (15% and 40%) were negative. In in vitro
micronucleus and chromosomal aberration assay positive results with olive extract or
hydroxytyrosol were reported. In the in vivo studies olive extract containing 15% hydroxytyrosol
did not induce micronuclei in rat bone marrow after four weeks of dosing at levels up to 561 mg
hydroxytyrosol/kg bw/day, while in a 90-day repeat-dose study, olive extract containing 35%
hydroxytyrosol produced increased rat bone marrow micronucleus frequencies, but the results
were questionable for various reasons. In acute micronucleus studies negative results were
obtained. A weight of evidence analysis of the in vitro and in vivo genotoxicity data for olive
extracts in general, for the specific olive extract (the subject of present GRAS assessment), and
the main olive polyphenol (hydroxytyrosol), demonstrates that any genotoxic risks for human
consumers are negligible (Kirkland et al., 2015).
6.2.2.3. Acute Toxicity Study
In addition to the above described specific acute toxicity studies with Hidrox®, in an
acute toxicity study, in a single dose toxicity study, D'Angelo et al. (200 1 ) investigated the acute
effects of hydroxytyrosol in rats. In this study, Sprague Dawley male rats (n=6) were treated with
a single oral (gavage) dose of 2000 mg hydroxytyrosol/kg bw. After the treatment, the rats were
observed for clinical signs. On day 14, the rats were euthanized and gross and pathological
changes in "main organs" (not specified in the publication) were evaluated. No deaths were
noted during the course of the study period. The only clinical sign observed in the rats was
piloerection, which started two hours after treatment and disappeared within 48 hours of
treatment. The findings from this study suggest that LDso ofehydroxytyrosol is >2000 mg/kg bw.
6.2.2.4. Absorption, Distribution, Metabolism and Excretion
In multiple publicly available publications on animal and human studies, the
bioavailability and metabolism of hydroxytyrosol (pure) or as a component of olive phenolics
present in olive oil or other olive-derived products (fruits, olive extracts, olive cake, etc.) has

Oliphenol Page 34 of 53 Hidrox®-GRAS

been extensively investigated. In several review articles, an overview on these in vitro and in
vivo animal and human studies on absorption, distribution, metabolism and excretion (ADME) of
olive phenolic compounds, with primary focus on hydroxytyrosol is provided (Soni et al., 2006;
Beck, 20 1 4 ; EFSA, 20 17; Karkovic Markovic et. al., 201 9).
The available information related to the bioavailability indicate that hydroxytyrosol as a
pure substance or as a component of olive oil or olive extracts is rapidly and dose-dependently
absorbed in humans and the rat (Bai et al., 1 998; Christian et al., 2004; Visioli et al., 2000, 200 1 ;
Miro-Casas et al., 200 1 , 2003; Tuck and Hayball, 2002; Covas et al., 2006; Soni et al., 2006;
Gonzalez-Santiago et al., 20 10; Kotronoulas et al., 201 3). Following absorption, hydroxytyrosol
is rapidly distributed in several tissues in rats, with no preference for a specific organ or tissue.
The available information shows a rapid decrease in plasma and tissue levels of hydroxytyrosol,
and there is no indication of any accumulation in the body (D'Angelo et al., 200 1 ; Serra et al.,
201 2).
The metabolism of hydroxytyrosol has been studied in some detail (Tuck et al., 200 1 ,
2002; D'Angelo et al., 200 1 ; Visioli et al., 2000, 2003; Caruso et al., 200 1 ; Vissers et al., 2002;
MiroCasas et al., 200 1 ; Rubio et al., 20 1 2a; Karkovic Markovic et. al., 20 1 9). A summary of
endogenous and exogenous metabolic pathways of hydroxytyrosol is presented in Figure 5. The
available information indicate that only a minor portion (<6%) of unchanged hydroxytyrosol is
found in plasma or urine, and the majority of hydroxytyrosol and its metabolites are present in
conjugated (glucuronides and sulfate) forms. The enzymes involved inHTyr phase-I metabolism,
mostly present in the intestinal wall, are non-microsomal alcohol and aldehyde dehydrogenases
(ALDH), both located in the cytosol. In addition to direct phase II conjugation ofthydroxytyrosol,
a major metabolic transformation is found to be the methylation (via catechol-o-methyl
transferase, COMT) leading to homovanillyl alcohol (HVAle) as well as to homovanillic acid.
The Intestinal phase II conjugation and COMT activity contribute to the high first pass
elimination observed. The enzymes involved in hydroxytyrosol phase-II reactions,
sulphotransferases (SULT), uridine 5'-diphosphoglucuronosyl transferases (UGT) and catechol­
O-methyltransferase (COMT), form the main hydroxytyrosol metabolites detected in biological
samples.
The bioavailability ofthydroxytyrosol in rats and humans following oral ingestion of olive
oil or other olive-derived food products (in humans), or administration of pure hydroxytyrosol
(in rats), has been investigated by several investigators. The findings from these studies reveal
the presence of hydroxytyrosol and its metabolites in blood and urine. Precursors of
hydroxytyrosol such as oleuropein and its aglycones are extensively hydrolyzed to
hydroxytyrosol in the gut (Corona et al., 2006; Pereira-Caro et al., 20 1 2; Mosele et al., 20 1 4) and
thus contribute to its high absorption (Visioli et al., 2003; Serra et al., 201 2; Kendall et al., 20 1 2).
Hydroxytyrosol is rapidly absorbed and reaches a plasma maximum within minutes (5-30 min)
after intake (Bai et al., 1 998; Miro-Casas et al., 2003; Gonzalez-Santiago et al., 20 10; Suarez et
al., 201 1 ; Rubio et al., 20 1 2b). Because of the high first pass metabolism in the intestine and
liver, elimination of hydroxytyrosol from plasma is also rapid. Hydroxytyrosol is primarily
excreted via urine and urinary excretion rate (including all metabolites) is highest within the first
8 hours (D'Angelo et al., 200 1 ; Tuck et al., 200 1 ; Visioli et al., 2000, 200t1 ; MiroCasas et al.,
200 1 ; Kountouri et al., 2007). These bioavailability estimates, based on recovery of
hydroxytyrosol and its metabolites in urine, reach levels >90% in rats (D'Angelo et al., 2001 ,

Oliphenol Page 35 of 53 Hidrox®-GRAS

Tuck et al., 2001), and range from 30-75% in human studies (Visioli et al., 2000; Vissers et al.,
2002; Miro-Casas et al., 2001; Weinbrenner et al., 2004a; 2004b).
0

~ OH
HO) l ) ~H2
Tyrosille HVA HVA-4'·0-sulpha!e

TH ! O
HO~OH HO~OH

HOµ ~HJ HO
~ 6
l·DOPA DOPAC
oocj ALDHl DOR
' MAO HO~H
HOrn
HO
I"" N-it HO~ 0
Oapamne OOPAL
. . . . .... ... . . ..... --......... :AI.R
(OdOfl()flOCJS SOCJIC.

l
ADH : lflllJO'I, _
• otlll EIOH
' ,ma>.o
~',./~\,O OH
HO.... ~ ~ -S: , UGT
t«) OH .~ ..,
HO HTyr 1-acetate-4'0-sulphate
Hlyr 3"..().glucU'Clrllde Hlyr

HO~OH
H~:-,../0 ~ •
HO~..,.,.,.-
_::_\_ ,O O s'O:cr-OH HO~OH
HO~ > I 03S, ~
HO ..,::: 0
Hlyr 4·.o.g1ucuronide Hlyr 3'.0-slAphate Hlyr 4'.0·SUIJ1,ate N-acety~S-S-cystern~ Hlyr

Figure 5. Metabolic Pathways of Endogenous and Exogenous Hydrox)1yrosol. HV Ale: homovanillic alcohol;
HVA: homovanillic acid; EtOH: ethanol; T H: tyrosine hydroxylase; DDC: dopa decarboxylase; MAO:
monoaminoxidase; ALDH: aldehyde dehydrogenase; ALR: aldehyde/aldosa reductase; ADH: alcohol
dehydrogenase; DOR : DOPAC reductase; COMT: catechol-O-methyltransferase; UGT: uridine 5'­
diphosphoglucuronosyl transferases; SULT: sulphotransferase; ACT: O-acetyltransferase; GGT: y-glutamyl
transpeptidase; NAT: N-acetyl transferase. (Adapted from Karkovic Markovic et. al., 2019)

6.2.2.5. Human Studies with Olive Preparations

In the published literature, several human clinical studies with olive oil and virgin olive
oil have appeared. Olive oil is a functional food that, besides its high content in mono­
unsaturated fatty acids (75%), also contains other minor, biologically active, components, such
as vitamins, minerals, and polyphenols. Virgin olive oil contain bioactive polyphenols as minor
components. Polyphenols, including hydroxytyrosol, as components of olive oil or olive leaf
extract has been investigated for their potential benefits in multiple clinical studies. In some
available review articles (Raederstorff, 2009; EFSA, 20 I 1; Rigacci and Stefani, 20 I 6; Tsartsou
et al., 2019), the available human clinical studies of olive polyphenols have been summarized. A
majority of the clinical studies of olive polyphenols are conducted to evaluate the efficacy. These
intervention studies suggest that olive polyphenols protects against oxidative damage as
evaluated by decreases the levels of oxidized-LDL in plasma. The available information suggest

Oliphenol Page 36 of53 Hidrox®-G RAS

that it is not feasible to achieve high dosages of olive polyphenols from consumption of olive oil.
The European Food Safety Authority (EFSA, 20 1 I ) has approved a health claim concerning the
effectiveness of the ingestion of olive oil polyphenols (5 mg/day) on protecting LDL from
oxidation.
In a randomized, crossover, controlled trial, Castaner et al. (2012) investigated the effects
of olive oil polyphenols on cardiovascular health benefits. In this study, 18 healthy European
volunteers who daily received 25 mL olive oil with a low polyphenol content of 2.7 mg/L or a
high polyphenol content of 366 mg/L in intervention periods of 3 weeks separated by 2 week
washout periods. The high polyphenol content group was associated with increased tyrosol and
hydroxytyrosol in urine and showed beneficial biomarker changes. The polyphenol intake from
high polyphenol content of olive oil was 9. 1 5 mg/day (366 mg/L x 25 ml/day) for an individual
weighing 60 kg. Compliance by participants was reported as good with no mention of adverse
effects. The polyphenol intake in this study can be considered as moderate.
In a another double-blinded, placebo-controlled, crossover trial, de Bock et al. (2013)
assessed the effects of olive leaf polyphenols (5 1 . 1 mg oleuropein, 9.7 mg hydroxytyrosol/day)
on insulin action and cardiovascular risk factors in middle-aged overweight male subjects. In this
study, 46 participants (aged 46.4±5.5 years and BM! 28.0±2.0 kg/m2) were randomized to
receive capsules with olive leaf extract or placebo for 1 2 weeks, crossing over to other treatment
after a 6-week washout. All participants took >96% of prescribed capsules. The extract
supplementation was associated with a 1 5% improvement in insulin sensitivity compared to
placebo. There was also a 28% improvement in pancreatic P-cell responsiveness. The extract
supplementation also led to increased fasting interleukin-6, IGFBP- 1 , and IGFBP-2
concentrations. There were, however, no effects on interleukin-8, TNF-a, ultra-sensitive CRP,
lipid profile, ambulatory blood pressure, body composition, carotid intima-media thickness, or
liver function. The results of this study revealed that supplementation with olive leaftpolyphenols
for 1 2 weeks significantly improved insulin sensitivity and pancreatic P-cell secretory capacity in
overweight middle-aged men at risk of developing the metabolic syndrome. The only adverse
event reported by one participant was a flare up of acne. This participant withdrew from the
study and un-blinding showed that he was receiving placebo. Liver function tests showed no
differences in AST, ALP, ALT, or GGT among participants in supplement vs placebo group.
In addition to the above described human studies with olive and its preparations, in the
published literature, some studies with hydroxytyrosol, the active constituent of olives, have
appeared. Crespo et al. (2015) tested the effects of hydroxytyrosol on expression of Phase II
enzymes in 2 1 healthy human subjects. In this double-blind, randomized, placebo controlled trial,
the effects of hydroxytyrosol were investigated following a Latin square design. After one-week
initial washout (i.e., olive-free diet), the subjects were randomly assigned to either the placebo
(maltodextrin), 5 mg hydroxytyrosol/day or 25 mg hydroxytyrosol/day groups. Administration of
each treatment was carried out for one week, followed by one-week washout after which
treatments were switched. In this study, Hytolive®, an olive mill and olive mill waste water
extract selectively enriched in hydroxytyrosol was used. Both 5 and 25 mg/day doses were well
tolerated and no adverse effects were reported. No differences in anthropometric variables or
significant variations in vital signs were noted.Hydroxytyrosol was well tolerated without any
significant alterations in Phase II enzyme expression in peripheral blood mononuclear cells.
In another randomized double-blinded, placebo-controlled crossover trial, Colica et al.
(201 7) determined the effect of hydroxytyrosol. In this study, healthy volunteers received two

Oliphenol Page 37 of 53 Hidrox®-GRAS

gastro-resistant capsules containing 15 mg hydroxytyrosol/day for 3 weeks. Evaluation of the
nutritional status, serum metabolites, oxidative stress biomarkers, and gene expression of 9 genes
related to oxidative stress, inflammation, and CVDs was perfonned. This study did not report
any adverse effect.
Lopez-Huertas and Fonolla ( 2017) investigated the effects of pure hydroxytyrosol in
humans as a supplement in an aqueous solution. In this study, hydroxytyrosol was administered
at a dose level of 45 mg/day for eight weeks to volunteers with mild hyperlipidemia ( n= l 4) and
markers of cardiovascular disease risk, enzyme markers of several clinical conditions,
hematology, antioxidant parameters, vitamins and minerals were measured at baseline, 4 weeks
and 8 weeks. The hydroxytyrosol dose administered was well tolerated, safe, and did not
influence markers of cardiovascular disease, blood lipids, inflammatory markers, liver or kidney
functions and the electrolyte balance in healthy subjects with borderline high levels of
cholesterol. Some minor changes were detected in biochemical parameters analyzed in serum
such as a decrease in lactate dehydrogenase or an increase in creatinine phosphokinase enzymes,
but their values were within the normal range without any clinical relevance. Serum iron levels
remained constant but a significant decrease in ferritin at weeks 4 and 8 was found albeit within
the physiological range. Serum folate and red blood cell folate levels were also reduced at weeks
4 and 8, while vitamin C increased by two-fold at weeks 4 and 8 as compared with levels at
baseline.
6.2.3. Secondary Unpublished Studies of Olive Extracts
6.2.3.1. Secondary Unpublished Toxicity Studies of Olive Extracts
Several unpublished studies of olive extracts, as well hydroxytyrosol, are described in the
GRAS notice ( GRN 726) dossier submitted by DSM ( 20 1 7). These studies provides further
support for the safe uses of olive extract and are briefly summarized here for the sake of
completeness. A summary of acute and short-term studies is provided in Table 1 1.

Table 1 1. Summar of Acute and Short-term Un ublished Toxicity Studies

StudV t e Route; Duration; Doses Results
Acute stud with olive extract from rocess used to make H40 H35
Mouse acute Oral (gavage); Single dose, 1 4 days; 5, 50, 300 or 2000 LD50 > 2000 mg/kg pilot plant
( ilot _
1------___,JL.......&,;__----<. !ant_extract)
________________ ~

extract 2:: 13 mg/kg hydroxytyrnsul

Short-term studies with Olive Extracts H drox tyrosol 15% S D
Rat Gavage; 2 weeks; I 0/sex/group 1 500 and 3000 1 5% 450 mg/kg bw/day high dose in
reliminar h drox t rosol formulation in feed and b ava e feed and b 0 avag e well tolerated
Rat sub-acute Gavage; 4 weeks; 5/sex/group, plus recovery animals, 0, 0 561 mg/kg bw/day
(placebo), 333, 1 000 and 3000, DSM 1 5% hydroxytyrosol
extract (0, 0, 62, 187 and 561 asehy drox t rosol)
GRN 726 Adapted from

The available unpublished studies of mutagenicity and genotoxicity described in GRN

726 ( DSM, 2017) are provided in Table 12.

,
Oliphenol Page 38 of 53 Hidrox®-GRAS
 . . Stu d'1es
T abl e 12 / 11 I•1tro M utat?emc1 t\' an dG enotox1c1ty
Hydroxytyro~I
Test Test System; Strain(s)/Target cells Results
concentration/ dose
Other Studies with H,·droxvtvrosol from Different Sources
S. typhi11111ri11111 (plate incorporation
Ames test with Both up to 5000 µg/plate in
and pre-incubation methods);
hydroxytyrosol 15% the presence and absence of Non-mutagenic
TA98, T AI00, TAdl535 , T Al537 and
SD metabolic activation
TA102
Hydroxytyrosol 15% Without S9: up to 200 µg/ml Positive or
CHO cells; With/without metabolic
SD With S9: Up to I 000 ~1g/ml borderline in
activation
MNT screening assay absence of S9
Positive or
Pure hydroxytyrosol CHO cells; With/without metabolic With and without S9: Up to
borderline in
MNT screening assay activation 200 ~1g/mL
absence of S9
Study with H35
Positive in
Without S9: 0.002 to 0.200
CHO cells; With/without metabolic absence of S9
MNT screening assay ~1g/ml; With S9: 0.039 to
activation Equivocal. in
5.000 µg/ml
presence of S9

The available unpublished reproduction toxicity study of 15% hydroxytyrosol olive

formulation (hydroxytyrosol 15% SD) described in GRN 726 (DSM , 2017) are provided in Table
13.
.. y Stu d y with Hyd rox vtyroso
T abl e 13. Summary o f Repro duction Tox1c1t - 0
Results; NOAEL for
Study type Route; Duration Doses (mg/kg bw/day)
hydroxytvrosol
Rat Gavage; Day 6 0, 333, I 000, and 3000 168 mg/kg bw/day
developmental through 20 of hydroxytyrosol 15% SD (0. 56, 168 (intermediate dosage)
toxicity eestation and 504. hydroxytyrosol)

6.2.3.1. Secondary Unpublished Human Studies of Olive Extracts

In an unpublished human study described in GRN 726 GRAS dossier (DSM, 2017),
volunteers were administered Hidrox at 400 mg/day and 800 mg/day, or approximately 8 and 16
mg/day in terms of doses of hydroxytyrosol (total dose split between a.m. and p.m.), over two
weeks. The findings showed that supplementation with hydroxytyrosol resulted in a significant
increase in plasma total antioxidant capacity, and there was an up regulation of the glutathione
defense system in skeletal muscle following strenuous exercise. No adverse effects or side
effects attributable to Hidrox were seen. Additional details of the study were not available. It
should be noted that this study described in GRN 726 is conducted with the product that is the
subject of this present GRAS.
In another unpublished study, also mentioned in GRN 726, the effects of 15%
hydroxytyrosol olive formulation (Hydroxytyrosol 15% SD) were investigated in a placebo­
controlled, double blind, parallel, cross-over clinical study. This 6-week clinical study used
dosages of 50 and 150 mg/day in terms ofhydroxytyrosol given orally to 19 to 22 young men per
group. There were no serious adverse events. There were four adverse events (out of 43 across
all groups) ascribed by the physician to the treatment: Platelet cell decrease in one subject at high
dose (already low at baseline); Tightness in chest in one subject at both high dose and low dose;
Mood swings in one subject at low dose and; Two further adverse events were a persistent cough
possibly linked to a respiratory infection. It is concluded that the study showed no safety
concerns for hydroxytyrosol at a dosage of 150 mg/ day orally to young men over six weeks. In

Oliphenol Page 39 of 53 Hidrox®-GRAS

GRN 726, it is stated that this study supports an ADI of 150 mg/day, in terms of hydroxytyrosol,
as derived from the 90-day rat safety study with H35. Additional details of the study were not
available for independent review.
6.2.4. Corroborative Information
6.2.4.1. FDA GRAS Notices on Olive Phenolic Preparation and Hydroxytyrosol
The FDA received three GRAS notifications on ingredients related to olive, one on
phenolic preparation from olive fruit [GRN 726 ( DSM, 2017)] and two on purified
hydroxytyrosol, an active constituent found in olive fruits [GRN 876 ( Nova Mentis, 20 1 9) and
GRN 600 ( Seprox, 2015)]. In these submissions, extensive data from the published literature on
phenolics from olives, including hydroxytyrosol, were presented by the notifiers. The FDA did
not question the acceptability and suitability of the available evidence to support the safe use of
olive phenolics and hydroxytyrosol as evidenced by the FDA 'no question' letters that were sent
to the notifiers. The discussion presented below suggests that the agency is comfortable with the
GRAS status of phenolic preparation from olive fruit and its highly pure active ingredient
hydroxytyrosol, for uses in selected foods as presented in these GRAS notices. As the subject of
this present GRAS, this assessment is substantially similar to the products or active constituent
of these FDA notifications, and, therefore, the studies described in these notifications can also be
utilized to support the safety in the present GRAS assessment of Aqueous Olive Pulp Extract
(Hidrox®). Although there are some differences in the compositional analysis, the available
information, particularly from a safety assessment perspective, indicates that the primary active
chemical in all these GRAS notices is identical and handled similarly in the body. A summary of
product similarities between the FDA notified ingredient and the subject of the present GRAS
evaluation is presented in Table 1 4.

Table 1 4 Companson of A,queous or1ve PUlp . h s·1m1·1 ar

I E xtrac t wit . t FDA GRAS Notices .
ngre d 1en
Specifications/ Hidrox elaVidaTM Hydroxytyrosol Hydroxytyrosol
Parameters (present GRAS) (GRN 726)* (GRN 876)* (GRN 600)*
Purple brownish Yellow - brown Yellow
Description Off white powder
powder Viscous liquid Viscous liquid
Recombinante£. Chemical
Source material Olive fruits Olive fruits
Coli synthesis
8% (minor
Phenolics 6% NA NA
polyphenols)
Hydroxytyrosol (HT) 3.5% 40% 99% 99%
Moisture 2.8 ( 1 -3)% 33-37% <0.5% <4%
Ash 9 . 6 (6-10)% 3% NA NA
Protein 6.7 (4-7)% 0.7-0.8% NA NA
Fat 27.0 (20-30)% 0.e1 % NA NA
Carbohydrate (total) 53.5 (40-55)% 1 7- 1 8% NA NA
Intended uses Multiple foods Multiple foods Multiple foods Multiple foods
5-10 mg HT per 5-1 0 mg HT per 5-1 0 mg HT per 5-1 0 mg HT per
Use levels
servin_g serving serving serving
52 mg HT/p/day 52 mg HT /p/day 52 mg HT/p/day 5 1 mg HT/p/day
EDI- cumulative (0.9 mg/kg (0.9 mg/kg (0.9 mg/kg (0.85 mg/kg
bw/day) bw/day) bw/day) bw/day)
Proposed use Proposed use Proposed use Proposed use
ADI
levels levels levels levels
Totality of Totality of Totality of Totality of
Safety determination
available evidence available evidence available evidence available evidence

Oliphenol Page 40 of 53 Hidrox®-G RAS

 *Adapted from GRN 726, GRN 876 and GRN 600; HT-Hydroxytyrosol; p-person; NA-Not available; ND -Not
detected

6.2.4.1.1. GRN 726- Phenolic Preparation from Olive Fruit

In 2017, DSM Nutritional Products, LLC ( DSM) submitted a GRAS notice ( GRN 726)
on phenolic preparation from olive fruit ( PPOF) for use as an ingredient and as an antioxidant in
bakery products; beverages; dairy products and substitutes; desserts; fats and oils; fruit juices and
nectars; dry seasoning mixes for meat, poultry, and fish; chewing gum; sauces, dips, gravies, and
condiments; snacks; and vegetable juices at levels of 5 to I O mg of hydroxytyrosol per serving of
food. PPOF is described as a clear, colorless liquid consisting oft2:40% hydroxytyrosol, which is
the major phenolic compound found in olives. PPOF was produced either by extraction of olive
fruit pomace or isolation from the water inherent in the olives ( the vegetation water). The food
grade specifications for PPOF included hydroxytyrosol content ( 2:40%), limits on ash ( <3%),
minor polyphenols ( <8%), and heavy metals and microbial contaminants. The cumulative
maximum ( 90th percentile) dietary exposure to hydroxytyrosol for the total users only U.S.
population ( 2 years and older) was determined as 52 mg/person/day ( 0. 9 mg/kg bw/day).
In this GRAS notification, DSM ( 20 1 7) extensively summarized and discussed the
published safety data ( for the period through August 2017) and information pertaining to olive
extracts containing up to 35% hydroxytyrosol as well as with pure hydroxytyrosol to support the
safety of PPOF. DSM summarized the published rat and human studies on the absorption,
distribution, metabolism, and excretion of hydroxytyrosol itself, as a component of olive oil or
olive-derived products. Several published acute oral toxicity studies in rodents administered by
gavage either pure hydroxytyrosol or olive extracts containing different amounts of
hydroxytyrosol were described, concluding that the LDso for hydroxytyrosol is >2000 mg/kg bw,
the highest dose tested. DSM stated that no adverse toxicological effects were reported in a
published subchronic 90-day oral toxicity study in rats, administered by gavage, olive extract
containing 35% hydroxytyrosol at doses up to 6 91 mg/kg bw/day ( equivalent to 250 mg
hydroxytyrosol/kg bw/day). Additionally, published reproductive toxicity and teratogenicity
studies in rats, administered by gavage, a hydrolyzed aqueous olive pulp extract containing 2.4%
hydroxytyrosol from days 6 to 20 of gestation were described. No adverse maternal, reproductive,
or developmental effects were reported for olive pulp extract at doses up to 2000 mg/kg bw/day
( equivalent to 4 8 mg hydroxytyrosol/kg bw/day), the highest dose tested.
Furthermore, findings from published human studies with either pure hydroxytyrosol or
olive extract containing 15% hydroxytyrosol for durations up to eight weeks were described.
Based on these studies, DSM concluded that no adverse effects were noted in any of these
human studies. The results of published in vitro and in vivo genotoxicity studies on
hydroxytyrosol and olive extracts tested indicate that any genotoxic risks for human consumers
are negligible. Based on the totality of the data and information described in the GRAS
notification, DSM concluded that PPOF is GRAS for its intended uses in food. Following the
review of the information summarized in GRN 726, as well as other information available to the
FDA, the agency provided a "no questions" letter to the notifier regarding the GRAS status of
PPOF under the intended conditions of use. Given the similarity of the product described in GRN
726 and the subject of present GRAS, the data and information described in GRN 726 are
applicable to the present GRAS.

Oliphenol Page 41 of053 Hidrox®-GRAS

6.2.4.1.2. GRN 600 and 876- Hydroxytyrosol
In these two GRAS notices [GRN 600 ( Seprox, 2015) and GRN 876 ( Nova Mentis,
2019)], extensive information on safety ofthydroxytyrosol, as well as olive preparations has been
summarized. In the first GRAS notice ( GRN 600), Seprox discussed published subchronic
toxicological studies in rats fed hydroxytyrosol, olive extract, or an aqueous olive pulp extract
containing hydroxytyrosol. In these studies, no adverse toxicological effects were repo1ied at
levels up to 250 mg hydroxytyrosol/kg bw/day. In this GRAS notice, Seprox summarized
published reproductive toxicity and teratogenicity studies in rats fed aqueous olive pulp extract.
N o adverse reproductive, maternal, or developmental effects were noted at 48 mg
hydroxytyrosol/kg bw/day, the highest level tested. Additionally, Seprox also summarized
published human studies ranging from one day to eight weeks duration with olive mill water
enriched with hydroxytyrosol, olive oil, high oleic sunflower oil, or olive phenolic concentrate
containing hydroxytyrosol. The hydroxytyrosol levels in these studies ranged from 2 to 97
mg/person/day. No adverse toxicological outcomes were noted in any of these human studies.
Furthermore, Seprox also discussed published in vitro and in vivo genotoxicity studies and
concluded that hydroxytyrosol is not genotoxic. Following the review of the information
summarized in GRN 600, the agency provided a "no questions'' letter to the notifier regarding
the GRAS status of hydroxytyrosol.
In the second, and more recent, GRAS notice on hydroxytyrosol ( GRN 876), Nova
Mentis ( 2019) also extensively reviewed and summarized safety studies of hydroxytyrosol and
olive preparations. The subject of this notice is the phenolic compound hydroxytyrosol (>99%
pure) produced by fermentation of a culture of non-pathogenic Escherichia coli BL2 1 ( DE3)
#145 strain. The studies summarized in this GRAS notice to support the safety of hydroxytyrosol
were similar to those described in GRN 726 and GRN 600. Nova Mentis ( 2019) discussed
published data and information supporting the safety of hydroxytyrosol based on a scientific
literature search conducted through April 2019.
Nova Mentis ( 2019) summarized the results of three published subchronic studies in rats.
In all three studies, the test substance was administered by gavage. In one study, no adverse
effects were observed at 50 mg/kg bw/day of hydroxytyrosol. In another study, no adverse
effects were reported after the administration of 691 mg olive extract containing 35%
hydroxytyrosol, equivalent to 250 mg/kg bw/day of hydroxytyrosol. In a third study, no adverse
effects were observed after the administration of 2000 mg/kg bw/day of olive pulp extract
(Hidrox®) estimated to provide 4 8 mg/kg bw/day of hydroxytyrosol. Based on the results of a
published reproductive toxicity study and a published developmental toxicity study in rats, Nova
Mentis ( 2019) concluded that olive extract containing hydroxytyrosol as its major component is
unlikely to be a reproductive toxicant and that no maternal or developmental toxicity was
reported at up to 2000 mg/kg bw/day of the extract ( or 4 8 mg/kg bw/day of hydroxytyrosol).
Based on the results of multiple published in vitro bacterial reverse mutation assays, in vitro
chromosomal aberration assays, and in vivo micronucleus assays, Nova Mentis ( 2019) concluded
that hydroxytyrosol is unlikely to be genotoxic or clastogenic. Nova Mentis ( 2019) also stated
that the results of human studies with olive oil containing phenolics, including hydroxytyrosol,
did not reveal any adverse effects. Following the review of the information summarized in GRN
876, the FDA provided a ··no questions" letter to Nova Mentis regarding the GRAS status of
hydroxytyrosol.

Oliphenol Page 42 of 53 Hidrox®-GRAS

6.2.4.2. Evaluation by the European Food Safety Authority (EFSA)
The EFSA (20 I I ) has issued a scientific opinion on health claims as it relates to dietary
intake of hydroxytyrosol and related polyphenol compounds from olive fruit and oil and
protection of blood lipids from oxidative damage. The EFSA panel critically reviewed the
available information and concluded that a cause-and-effect relationship has been established
between the consumption of hydroxytyrosol and related compounds from olives and olive oil and
protection of blood lipids from oxidative damage. The EFSA panel determined that a minimum
of 5 mg of hydroxytyrosol and its derivatives in olive oil should be consumed daily to use a
cardiovascular health claim. Although, the EFSA panel did not comment on the safety of
hydroxytyrosol, it can be assumed that this ingredient is safe for human consumption at the
recommended level.

6.7. Expert Panel Review, Summary and Discussion

At the request of Oliphenol LLC, an independent panel of recognized experts (hereinafter

referred to as the Expert Panel)2 , qualified by their scientific training and relevant national and
international experience to evaluate the safety of food and food ingredients, was convened to
evaluate the Generally Recognized As Safe (GRAS) status of Aqueous Olive Pulp Extract
(Hidrox®), for use as a food ingredient and as an antioxidant in multiple selected food products,
described in this dossier, and at use levels to deliver 5 to I O mg of hydroxytyrosol per serving
(reference amounts customarily consumed, 21 CFR 101.12). A comprehensive search of the
scientific literature for safety and toxicity information on olive fruit, its preparations, olive
phenolics and active constituent such as hydroxytyrosol was conducted through August 2020 and
made available to the Expert Panel. The Expert Panel independently and critically evaluated
materials submitted by Oliphenol and other information deemed appropriate or necessary.
Following an independent, critical evaluation, the Expert Panel conferred on October 16, 2020
and unanimously agreed to the decision described herein.
Oliphenol ensured that all reasonable efforts were made to identify and select a balanced
Expert Panel with expertise in food safety, toxicology, and nutrition. The Expert Panel was
selected and convened in accordance with the Food and Drug Administration (FDA)'s guidance
for industry on "Best Practices for Convening a GRAS Panel" 3 • Efforts were placed on
identifying conflicts of interest or relevant "appearance issues" that could potentially bias the
outcome of the deliberations of the Expert Panel and no such conflicts of interest or "appearance
issues" were identified. The Expert Panel members received a reasonable honorarium as
compensation for their time; the honoraria provided to the Expert Panel members were not
contingent upon the outcome of their deliberations.
The Olea europaea L. plant bear a well-known fruit that is a berry, capsule, drupe (e.g.,
Olea, olive). The fruit is initially green then red and blue-black when ripe, surrounds a very hard
stone (or pit), which contains oblong compact seeds with plentiful endosperm. This fruit has long
been recognized as having inherent nutritional and health-enhancing potential. Despite its known
nutritional value, raw olives are rarely consumed and the fruit undergoes extensive processing to
produce the forms most commonly consumed, i.e., table olives and olive oil. The oil contained in

2Modeled after that described in section 20 l (s) of the Federal Food, Drug, and Cosmetic Act, As Amended. See also
attachments (curriculum vitae) documenting the expertise ofthe Panel members.
3 Available at: https://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatorylnformation/ucm583856.htm

Oliphenol Page 43 of 53 Hidrox®-GRAS

olives is normally extracted by a multi-stage process. Preparation of edible olives involves
pickling in a solution of lye to remove the bitter taste (rendered by oleuropein), and this practice
has been in use since Roman times. The content of phenolic compounds (simple as well as
complex) in olives and olive oil that are considered as important constituents depends on the
cultivars and the ripeness of the fruit at the time of harvest. Oliphenol has developed and
standardized a method to produce Aqueous Olive Pulp Extract (Hidrox®) from byproducts of
olive oil processing by he water extraction process. The extract is produced as standardized
powder and liquid forms.
The Aqueous Olive Pulp Extract (Hidrox®) is a standardized powder and liquid. The
preparations (powder and liquid) have an odor of processed olives and a characteristic aromatic
sour/olive flavor. The biologically active constituents of Hidrox® are polyphenols. Among the
phenolics, the major constituent of the pulp extract is hydroxytyrosol (50-70%), while other
polyphenols present include oleuropein (5-1 0%), tyrosol (0.3%), oleuropein aglycone and gallic
acid. All of the polyphenols present in the extract are also found in olive oil and are thus
commonly consumed. The phenolic acid content of the extract is 6%, while the hydroxytyrosol
content is approximately 2.0-3.5%. As a food ingredient, Aqueous Olive Pulp Extract (Hidrox®)
will be added to bakery products; beverages; dairy products and substitutes; desserts; fats and
oils; fruit juices and nectars; dry seasoning mixes for meat, poultry and fish; chewing gum;
sauces, dips, gravies and condiments; snacks; and vegetable juices, such that it will deliver 5 to
1 0 mg of hydroxytyrosol per serving (reference amounts customarily consumed, 2 1 CFR
1 0 1 . 1 2). Given that the extract contains 3.5% hydroxytyrosol, the per serving use levels of the
extract will be approximately 1 50 to 300 mg/serving. The proposed use of the extract will result
in a 90th percentile per user cumulative estimated daily intake of 52.4 mg/person/day (0.9 mg/kg
bw/day). The resulting 90th percentile intake of Aqueous Olive Pulp Extract (Hidrox®) will be
approximately 1 500 mg/person/day (25 mg/kg bw/day).
In a series of specifically designed studies, the potential toxicity of Aqueous Olive Pulp
Extract (Hidrox®) has been extensively investigated. These pivotal studies included Subchronic
toxicity, Reproductive/Developmental Toxicity, Developmental Toxicity, Short-term toxicity,
Acute toxicity and Genotoxicity (Ames assay; in vitro chromosomal aberration assay and in vivo
micronucleus assay). All of these pivotal studies were published in the peer-reviewed scientific
literature (Christian et al., 2004; Soni et al., 2006) and thus meet the requirement for the
"common knowledge" element of GRAS assessment. In the acute toxicity studies in rats and
mice, the LDso of extract was > 2000 mg/kg. In a short term study, oral administration of the
extract to rats at dose levels of 5000 mg/kg bw/day for 29 days, no mortality or clinical signs of
toxicity were noted, suggesting the LDso to be greater than 5000 mg/kg.
In the subchronic study in rats, the gavage administration of an aqueous pulp extract at
doses up to 2000 mg/kg/day for a period of 90 days did not reveal any signs of toxicity. Markers
of liver function tests, such as levels of ALT, AST and SDH, trended downward. However, the
decreases were within historical control values ranges and do not represent treatment-related
toxicity. Histological investigations of the major tissues did not reveal any treatment-related
pathological changes except for very low to mild focal hyperplasia of the mucosa! squamous
epithelium of the limiting ridge of the forestomach, apparently related to gavage administration
of the extract and the granularity and high viscosity of the suspended extract. Based on the data
from this subchronic toxicity study, the no observed adverse effect level (NOAEL) of the extract
for rats is 2000 mg/kg/day, the highest dose tested.

Oliphenol Page 44 of 53 Hidrox®-GRAS

 In a developmental toxicity study in rats, Aqueous Olive Pulp Extract (Hidrox®) did not
cause maternal or developmental toxicity at levels up to 2000 mg/kg/day (highest dose tested). In
an oral dose-range reproduction study in rats, doses of the extract ranging from 500 to 2000
mg/kg/day did not adversely affect any of the parental reproductive performance parameters
( estrous cycling, mating, fertility, parturition, lactation, maternal behavior) investigated or the
viability, growth or development of the offspring through one week postpartum. In an in vitro
mutagenicity study, aqueous olive pulp extract was not mutagenic in the presence or absence of
metabolic activation in S. typhimurium strains TA97 and TA 1535, while the results in strains
TA98 and TAI 00 were equivocal. In genotoxicity assays with E. coli, no mutagenicity was noted.
In chromosome aberration studies in Chinese hamster ovary cells, the extract elicited increases in
aberrant cells at I 000 µg/ml in the presence of metabolic activation. In contrast to these in vitro
positive results, in an in vivo micronucleus assay, exposure of rats to the extract did not induce
increases in polychromatic erythrocytes in bone marrow.
The NOAEL in the subchronic ( 90-day) toxicity, reproductive/developmental toxicity
and developmental toxicity studies was the highest level tested, 2000 mg/kg bw/day. As
compared to the NOAEL of 2000 mg/kg bw/day determined from the subchronic toxicity study
(the highest dose tested), the maximum daily intake of 25 mg/kg bw/day of Aqueous Olive Pulp
Extract (Hidrox®) from its proposed food uses is over 80-fold lower. Although this safety
margin is lower as compared to the standard 100-fold, the additional studies as described in this
dossier, and also mentioned below, further provides support for the safe uses of Aqueous Olive
Pulp Extract (Hidrox®) at the proposed use levels.
In addition to the above described pivotal studies of Aqueous Olive Pulp Extract
(Hidrox®), the safety of the extract is supported by several secondary published and unpublished
studies, as well as by corroborative evidence. These studies include the safety studies with other
olive extracts, including higher concentrated preparations (containing 35 and 1 5%
hydroxytyrosol), and studies with pure hydroxytyrosol that are summarized in this dossier. The
two published subchronic toxicity studies include one in which no adverse effects were reported
after the administration of 6 91 mg olive extract containing 35% hydroxytyrosol, equivalent to
250 mg/kg bw/day of hydroxytyrosol (Heilman et al., 201 5) and the other study in which, no
adverse effects were observed at 50 mg/kg bw/day of pure hydroxytyrosol (Aunon-Calles et al.,
2013a). Several published acute oral toxicity studies in rodents administered by gavage either
pure hydroxytyrosol or olive extracts containing different amounts of hydroxytyrosol suggest the
LDso for hydroxytyrosol is>2000 mg/kg bw, the highest dose tested.
A critical analysis of in vitro and in vivo genotoxicity data for olive extracts and the main
olive polyphenol (hydroxytyrosol) suggest that any genotoxic risks for human consumers are
negligible (Kirkland et al., 20 1 5). Additionally, published rat and human studies on the
absorption, distribution, metabolism, and excretion of hydroxytyrosol itself, as a component of
olive oil or olive-derived products, suggest that hydroxytyrosol is unlikely to accumulate in the
body. Furthermore, an unpublished study (described in GRN 726) in rats with olive extract
(Hydroxytyrosol 15% SD) provided a NOAEL of 168 mg/kg bw/day when expressed in terms of
hydroxytyrosol. Based on these secondary studies, application of a 100-fold safety factor to the
NOAEL (250 mg hydroxytyrosol/kg bw/day ) from the sub-chronic study with olive extract 35%
results in an ADI of 150 mg hydroxytyrosol/day (for a 60 kg person). Similarly, applying a 1 00-
fold safety factor to the NOAEL of 1 6 8 mg/kg bw/day from an embryo-fetal (developmental)
toxicity study gives an intake up to I 00 mg/day in terms of hydroxytyrosol for a 60 kg adult. All
of these studies suggest that exposure to 52 mg hydroxytyrosol/day from the proposed uses of

Oliphenol Page 45 of 53 Hidrox®-GRAS

Aqueous Olive Pulp Extract (Hidrox®), including background intake, is unlikely to cause any
adverse effects and is considered as safe.
In summary, there is sufficient qualitative and quantitative scientific evidence, including
animal data, to assess the safety-in-use for Aqueous Olive Pulp Extract (Hidrox®), the subject of
this present GRAS assessment. The safety assessment of Aqueous Olive Pulp Extract (Hidrox®)
is based on the totality of available evidence, including a variety of specifically designed animal
toxicity studies. The totality of the available evidence supports the safety of Aqueous Olive Pulp
Extract (Hidrox®) at the maximum (90th percentile) all users intake of 1 500 mg/person/day (25
mg/kg bw/day) and its active constituent hydroxytyrosol at maximum cumulative intake of 52
mg/person/day. On the basis of scientific procedures4, the consumption of Aqueous Olive Pulp
Extract (Hidrox®) as an added food ingredient is considered safe at use levels up to 300
mg/serving. The intended uses are compatible with current regulations, i.e., Aqueous Olive Pulp
Extract (Hidrox®) is used in specified foods (described in this document) and is produced
according to current good manufacturing practices (cGMP).

4
2 1 CFR § 170.3 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.

Oliphenol Page 46 of 53 Hidrox®-GRAS

6.8. Expert Panel Conclusion

Based on a critical evaluation of the publicly available data, summarized herein, the
Expert Panel members whose signatures appear below, have individually and collectively
concluded that Aqueous Olive Pulp Extract (Hidrox®), meeting the specifications cited herein,
and when used as a food ingredient and as an antioxidant at use levels ranging from 150 to 300
mg/serving ( used such that it will deliver 5 to 10 mg of hydroxytyrosol per serving) in
conventional foods such as bakery products; beverages; dairy products and substitutes; desserts;
fats and oils; fruit juices and nectars; dry seasoning mixes for meat, poultry and fish; chewing
gum; sauces, dips, gravies and condiments; snacks; and, vegetable juices ( when not otherwise
precluded by a Standard of Identity) as described in this monograph, and resulting in the
maximum ( 90th percentile) estimated intake of 1500 mg Aqueous Olive Pulp Extract
(Hidrox®)/person/day, is safe.

It is also our opinion 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 Aqueous Olive Pulp Extract (Hidrox®), when used as
described, is Generally Recognized As Safe ( GRAS) based on scientific procedures.

Signatures

Rohe;-L. Marli;-Plill -
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Date

1\ 1 h<i1mL'i. Ph.D .. I· .AC' .1 .. L\. f s.

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C)t-1. . .2 I J 202...0
\fadhusudan c ; Soni. Ph.D .. r .A.C. Dare
F.A.l.S. !\Jv 1t1r lo f 'JX.'rl Panel

Oliphenol Page 47 of53 Hidrox®-GRAS

Part VII- SUPPORTING DOCUMENTS AND REFERENCES

Angerosa, F.; d'Alessandro, N.; Corana, F., Mellerio, G. 1 996. Characterisation of phenolic and
secoiridoid aglycones present in virgin olive oil by gas chromatography-chemical
ionisation mass spectrometry. Journal of Chromatography 736:1 95-203.
Aufion-Calles, D., Canut, L., Visioli, F. 2013a. Toxicological evaluation of pure hydroxytyrosol.
Food and Chemical Toxicology 5 5 :4 98-504.
Aufion-Calles, D., Giordano, E., Bohnenberger, S., Visioli, F. 2013b. Hydroxytyrosol is not
genotoxic in vitro. Pharmacol. Res. 74:t87-93.
Bai, C., Yan, X., Takenaka, M., Sekiya, S., Nagata, T. 1998. Determination of synthetic
hydroxytyrosol in rat plasma by GC-MS. J Agric Food Chem 46:3 998-400 I .
Beck, M. 2014. Hydroxytyrosol ( CAS No. 105t97-60-1) Overview of published ADME data
( absorption, distribution, metabolism, excretion). Internal DSM RDR Report No
00023941.
Bitler, C., Matt, K., Irving, M., Hook, G., Yusen, J., Eagar, F., Kirschner, K., Walker, B., Crea, R.
2007. Olive extract supplement decreases pain and improves daily activities in adults
with osteoarthritis and decreases plasma homocysteine in those with rheumatoid arthritis.
Nutrition Research 27:470-477.
Blekas, G.; Vassilakis, C.; Harizanis, C.; Tsimidou, M., Boskou, D.G. 2002. Biophenols in table
olives. Journal of Agricultural and Food Chemistry 50:36 88-3692.
Borzillo, A.; Iannotta, N., Uccella, N. 2000. Oinotria table olives: Quality evaluation during
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Brenes-Balbuena, M.; Rejano, L.; Garcia, P.; Sanchez, A.H., Garrido, A. 1 995. Biochemical
changes in phenolic compounds during Spanish-style green olive processing. Journal of
Agricultural and Food Chemistry 43:2702-2706.
Brenes-Balbuena, M.; Garcia-Garcia, P., Garrido-Fernandez, A. 1 992a. Concentration of
phenolic compounds change in storage brines of ripe olives. Journal of Food Science
5 8:347-350.
Brenes-Balbuena, M.; Garcia-Garcia, P., Garrido-Fernandez, A. 1 992b. Phenolic compounds
related to the black colour formed during the processing of ripe olives. Journal of
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Caruso, D.; Visioli, F.; Patelli, R.; Galli, C., Galli, G. 2001. Urinary excretion of olive oil
phenols and their metabolites in humans. Metabolism 50: 1426-142 8.
Castaner, 0., Covas, M-I., Khymenets, 0., Nyyssonen, K., Konstantinidou, V., Zunft, H-F., de la
Torre, R., Munoz-Aguayo, D., Vila, J., Fito, M. 2012. Protection of LDL from oxidation
by olive oil polyphenols is associated with a down regulation of CD40-ligand expression
and its downstream products in vivo in humans. Am J Clin Nutr 95 :1238-1244.

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Christian, M.; Sharper, V.; Hoberman, A.; Seng, J.; Fu, L.; Covell, D.; Diener, R.; Bitler, C.,
Crea, R. 2004. The toxicity profile of hydrolyzed aqueous olive pulp extract. Drug and
Chemical Toxicology 27:309-330.
Colica, C., Di Renzo, L., Trombetta, D., Smeriglio, A., Bernardini, S., Cioccoloni, G., Costa de
Miranda, R., Gualtieri, P., Sinibaldi Salimei, P., De Lorenzo, A. 20 17. Antioxidant
effects of a Hydroxytyrosol-based pharmaceutical fonnulation on body composition,
metabolic state, and gene expression: A randomized double-blinded, placebo-controlled
crossover trial. Oxid. Med. Cell Longev. 2017:2473495.
Corona, G., Tzounis, X., Assunta Dessi, M., Deiana, M., Debnam, E.S., Visioli, F., Spencer, J.P.
2006. The fate of olive oil polyphenols in the gastrointestinal tract: Implications of gastric
and colonic microflora-dependent biotransformation. Free Radie Res 40(t 6):647-658.
Covas, M.I., de la Torre, K., Farre-Albaladejo, M., Kaikkonen, J., Fito, M., Lopez-Sabater, C.,
PujadasBastardes, M.A., Joglar, J., Weinbrenner, T., Lamuela-Raventos, R.M., de la
Torre, R. 2006. Postprandial LDL phenolic content and LDL oxidation are modulated by
olive oil phenolic compounds in humans. Free Radie Biol Med 40:608-616.
Crespo, M.C., Tome-Carneiro, J., Burgos-Ramos, E., Loria Kohen, V., Espinosa, M.I., Herranz,
J., Visioli, F. 2015. One-week administration of hydroxytyrosol to humans does not
activate Phase II enzymes. Pharmacol. Res. 95- 96 : 132- 137.
D'Angelo, S.; Manna, C.; Migliardi, V.; Mazzoni, O.; Morrica, P.; Capasso, G.; Pantoni, G.;
Galletti, P., Zappia, V. 200t1. Pharmacokinetics and metabolism of hydroxytyrosol, a
natural antioxidant from olive oil. Drug Metabolism and Disposition 29: 1492-1498.
De Bock, M., Derraik, J.G.B., Brennan, C.M., Biggs, J.B., Morgan, P.E., Hodgkinson, S.C.,
Hofman, P.L., Cutfield, W.S. 2013. Olive (Olea europaea L.) leaf polyphenols improve
insulin sensitivity in middle-aged overweight men: A randomized, placebo-controlled,
crossover trial. PLoS One 8(t 3):e5 7622. doi: 10. 1371/journal.pone.0057622.
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Marsilio, V.; Campestre, C., Lanza, B. 200 I . Phenolic compounds changes during California­
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Mosele, J.I., Martin-Pelaez, S., Macia, A., Farras, M., Valls, R.M., Catalan, U., Motilva, M.J.
20t14. Faecal microbial metabolism of olive oil phenolic compounds: In vitro and in vivo
approaches. Mo! Nutr Food Res. 58: 1809-1 819.
Nova Mentis, 2019. Hydroxytyrosol. GRN No.:t8 76. Complete GRAS notice available at:
https://www.fda.gov/media/134474/download
OECD Guidelines for the Testing of Chemicals. OECD 408, Repeated dose 90-day oral toxicity
study in rodents. Adopted: 21 September 1998 OECD Guidelines for the Testing of
Chemicals, OECD 541, Carcinogenicity Studies, Adopted: 7 September 200t9.
Oliphenol LLC. 2020. Information on General description, Identity, Specifications, Composition
and Manufacturing of Hidrox®. Unpublished.
Owen, R.W.; Haubner, R.; Mier, W.; Giacosa, A.; Hull, W.E.; Spiegelhalder, B., Ba1tsch, H.
2003. Isolation, structure elucidation and antioxidant potential of the major phenolic and
flavonoid compounds in brined olive drupes. Food and Chemical Toxicology 41 : 703-717.
Pereira-Caro, G., Sarria, B., Madrona, A., Espartero, J.L., Escuderos, M.E., Bravo, L., Mateos, R.
2012. Digestive stability of hydroxytyrosol, hydroxytyrosyl acetate and alkyl
hydroxytyrosyl ethers. Int J Food Sci Nutr 63:t703-707.
Raederstorff, D. 200t9. Antioxidant activity of olive polyphenols in humans: A review. Int. J.
Vitam. Nutr. Res. 7 9( 3):152-165.
Rigacci, S., Stefani, M. 2016. Nutraceutical properties of olive oil polyphenols. An itinerary
from cultured cells through animal models to humans. International J. Mole. Sci.
17( 6): 843. https://doi.org/10.33 90/ijms I 7060843t.
Robards, K.; Prenzler, P.D.; Tucker, G.; Swatsitang, P., Glover, W. 1 999. Phenolic compounds
and their role in oxidative process in fruits. Food Chemistry 66:40 1 -436.
Rodriguez-Lara, A., Mesa, M.D., Aragon-Vela, J., Casuso, R. A., Vazquez, C.C., Zuniga, J.M.,
Huertas, J.R. 2019. Acute/subacute and sub-chronic oral toxicity of a hidroxytyrosol-rich
virgin olive oil extract. Nutrients 1 1( 9):2133. https://doi.org/10.3390/nul 1092 133.
Romero, C.; Brenes, M.; Yousfi, K.; Garcia, P.; Garcia, A., Garrido, A. 2004. Effect of cultivar
and processing method on the contents of polyphenols in table olives. Journal of
Agricultural and Food Chemistry 52:479-484.

Oliphenol Page 51 of 53 Hidrox®-GRAS

Rubio, L., Macia, A., Valls, R.M., Pedret, A., Romero, M-P., Sola, R., Motilva, M-J. 20 1 2a. A
new hydroxytyrosol metabolite identified in human plasma: Hydroxytyrosol acetate
sulphate. Food Chem 1 34: 1 1 32-1 1 36.
Rubio, L., Valls, R.M., Macia, A., Pedret, A., Giralt, M., Romero, M.P., de la Torre, R., Covas,
M.I., Sola, R., Motilva, M.J. 20 1 2b. Impact of olive oil phenolic concentration on human
plasmatic phenolic metabolites. Food Chem 1 35(t4):2922-2929.
Ryan, D.; Robards, K., Lavee, S. 1 999. Changes in phenolic content of olive during maturation.
Journal of Food Science and Technology 34:265-274.
Sciancalepore, V., Longone, V. 1 9 84. Polyphenol oxidase activity and browning in green olives.
Journal of Agricultural and Food Chemistry 32:320-321.
Seprox Biotech S.L., 20 1 5. Hydroxytyrosol. Complete GRAS notice available at:
https://wayback.archive-
it.org/7993/2019020t8035755/https:/www.fda.gov/downloads/Food/IngredientsPackaging
Labeling/GRAS/Noticelnventory/ucm494696.pdf
Serra, A., Rubio, L., Borras, X., Macia, A., Romero, M.P., Motilva, M.J. 20 1 2. Distribution of
olive oil phenolic compounds in rat tissues after administration of a phenolic extract from
olive cake. Mol Nutr Food Res 56:4t86-496.
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aqueous olive pulp extract as an antioxidant or antimicrobial agent in foods. Food and
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Suarez, M., Valls, R.M., Romero, M.P., Macia, A., Fernandez, S., Giralt, M., Sola, R., Motilva,
M.J. 20 1 1 . Bioavailability of phenols from a phenol-enriched olive oil. Br J Nutr
1 06: 1 69 1 -170 1 .
Tennant, B.C. 1 999 Assessment of hepatic function. In The Clinical Chemistry of Laboratory
Animals. ( F. W. Quimby, Ed.). Taylor & Francis, Philadelphia, PA. p. 501-517.
Tsartsou, E., Proutsos, N., Castanas, E., Kampa, M. 2019. Network meta-analysis of metabolic
effects of olive-oil in humans shows the importance of olive oil consumption with
moderate polyphenol levels as part of the Mediterranean diet. Front. Nutr. 6:6.
https://doi.org/1t0.3389/fnut.20 1 9.00006.
Tuck, K.L.; Hayball, P.J., Stupans, I. 2002. Structural characterization of the metabolites of
hydroxytyrosol, the principal phenolic component in olive oil, in rats. Journal of
Agricultural and Food Chemistry 50:2404-2409.
Tuck, K.L., Hayball, P.J. 2002. Major phenolic compounds in olive oil: Metabolism and health
effects. The Journal of Nutritional Biochemistry 1 3:644.
Tuck, K.L.; Freeman, M.P.; Hayball, P.J.; Stretch, G.L., Stupans, I. 200 1 . The in vivo fate of
hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after
intravenous and oral dosing of labeled compounds to rats. Journal of Nutrition 1 3 1 : 1 993-
1996.
Visioli, F.; Galli, C.; Grande, S.; Colonnelli, K.; Patelli, C.; Galli, G., Caruso, D. 2003.
Hydroxytyrosol excretion differs between rats and humans and depends on the vehicle of
administration. Journal of Nutrition 1 33:26 1 2-2615.

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Visioli, F., Galli, C. 2003 Olives and their production waste products as sources of bioactive
compounds. Current Topics in Nutraceutical Research I :85-88.
Visioli, F.; Caruso, D.; Plasmati, E.; Patelli, R.; Mulinacci, N.; Romani, A.; Galli, G., Galli, C.
200 I . Hydroxytyrosol, as a component of olive mill waste water, is dose-dependently
absorbed and increases the antioxidant capacity of rat plasma. Free Radical Research
34:301 -305.
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in vitro and in vivo studies. World Review ofNutrition and Dietetics 88:233-237.
Visioli, F.; Galli, C.; Bomet, F.; Mattei, A.; Patelli, R.; Galli, G., Caruso, D. 2000. Olive oil
phenolics are dose-dependently absorbed in humans. FEBS Letters 468 : 1 59- 1 60.
Vissers, M.N., Zock, P.L., Roodenburg, A.J., Leenen, R., Katan, M.B. 2002. Olive oil phenols
are absorbed in humans. J Nutr 1 32:409-41 7.
Weinbrenner, T., Fito, M., de la Torre, R., Saez, G.T., Rijken, P., Tormos, C., Coolen, S.,
Albaladejo, M.F., Abanades, S., Schroder, H., Marrugat, J., Covas, M.l. 2004a. Olive oils
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de La Torre, R., Covas, M.l. 2004b. Bioavailability of phenolic compounds from olive oil
and oxidative/antioxidant status at postprandial state in healthy humans. Drugs Exp Clin
Res 30:207-2 12.

Oliphenol Page 53 of 53 Hidrox®-GRAS

Santos, Marissa

From: Madhu Soni <sonim@bellsouth.net>

Sent: Monday, July 12, 2021 3:55 PM
To: Santos, Marissa
Subject: [EXTERNAL] RE: GRN 000978 - Question for the Notifier
Attachments: GRN 978 FDA Query Responses final-1.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 Ms. Santos,

Please find attached a pdf file providing a point-by-point response to the agency queries related to our GRAS
notification (GRN 000978) .
I hope the information and clarifications, along with some discussion in the response addresses the FDA queries.
If you have any questions or need additional explanation, please let me know.
Thank you for the opportunity to provide this explanation.
Best regards
Madhu
------------------------------------------
Madhu Soni, PhD, FACN, FATS
Soni & Associates Inc.
749 46th Square
Vero Beach, FL 32968, USA
Phone: +1-772-299-0746
Cell: +1-772-538-0104
www.soniassociatesnet

From: Santos, Marissa [mailto:Marissa.Santos@fda.hhs.gov]

Sent: Friday, June 25, 2021 1:55 PM
To: Madhu Soni <sonim@bellsouth.net>
Subject: GRN 000978 ‐ Question for the Notifier

Dear Dr. Soni,

During our review of GRAS Notice No. 000978, we noted several questions that need to be addressed and are attached
to this email.

We respectfully request a response wi thin 10 business days. If you are unable to

complete the response within that time frame, please contact me to discuss further options. Please do not include any
confidential information in your responses.

If you have any questions or need further clarification, please feel free to reach out to me.

Regards,
Marissa

Marissa Santos, M.S.

1
Regulatory Review Scientist
Division of Food Ingredients
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
U.S Food and Drug Administration
Tel: 240.402.8160
marissa.santos@fda.hhs.gov
The link ed image cannot be display ed. The file may hav e been mov ed, renamed, or deleted. Verify that the link points to the correct file and location.

2
Dear Dr. Santos,

RE: GRN 978 (Hydrolyzed aqueous olive pulp extract)

This responds to your email of June 25, 2021, regarding your queries that need to be
addressed for Hydrolyzed aqueous olive pulp extract GRAS Notice (GRN 978) submitted on
behalf of Oliphenol LLC. We are providing a point-by-point response to all your queries along
with some additional relevant clarifications/discussion.

FDA Query: (1) In Table 3 (page 8) of your notice, you provide the specifications for aqueous
olive pulp extract and list the method used to assess water solubility as an “in house”
method. Please indicate the concentration of the solutions and the temperature at which
the test is conducted. Please also confirm that the method to determine water solubility is
validated for the intended purpose of assessing solubility of the two forms of aqueous olive
pulp extract.

Response: The “in house” method is based upon centrifugation. A 1% solution of the test
compound (HIDROX® powder) is stirred in water for 15 minutes at room temperature. The
solution is then centrifuged at 12,000 rpm for 15 minutes and the solid residue at the bottom of
the glass tube is separated from the supernatant, dried in an oven at 70ºC for 4 hours, and
weighed. The solubility is calculated as % difference between the original sample minus the
quantity of solid collected after centrifugation. We apply this standard assay to both HIDROX®
in powder and HIDROX® in liquid form. We confirm that the method to determine water
solubility is validated for the intended purpose of assessing solubility of the two forms of
aqueous olive pulp extract.

FDA Query: (2) On pages 10-12 of the notice you describe and provide a flow chart of the
manufacturing process for aqueous olive pulp extract. We request that the method of
manufacture is clarified.

a) Your narrative states that aqueous olive pulp extract is manufactured from the byproducts
of olive oil production and the flow chart indicates that olive pomace is the starting
material. Please comment on whether there is a pasteurization step and if the olive
enzymes are inactivated during the manufacture of aqueous olive pulp extract.

Response: a) Please note that there is no pasteurization step and or enzyme inactivation step in
the production of the olive pomace. The olive pulp is heated to 30ºC to facilitate the
separation of the olive oil and olive vegetation water. This temperature does not denature
proteins.

Page 1 of 8
 b) On page 12 you state the ‘manufacturing flow chart for liquid aqueous olive pulp extract is
provided below.’ However, there is no flow chart specifically for the manufacture of
liquid aqueous olive pulp extract after the text. If there is a figure missing or this is a
misstatement, please provide the figure or clarify for the record.

Response: b) Thank you for bringing this to our attention and sorry for the misstatement. The
Figure 3 flow chart shows the manufacturing flow for both the HIDROX® powder and the
HIDROX® liquid. In Figure 3, the “powder” process branches off to the left and the
“liquid” branches off to the right on the flow chart.

FDA Query: (3) In Table 1 you provide a general description of the characteristics of aqueous
olive pulp extract and list a shelf life of 2 years. Please provide information that
substantiates that the food ingredient has the indicated shelf life, including information
regarding the test substance (powder or liquid) and the indicator parameters measured.

Response: Aqueous olive pulp extract (HIDROX®) was tested in an accelerated and long-term
shelf-stability study on representative lots of HIDROX® 6% Freeze-dried Powder. Accelerated
shelf-life samples were stored at 40ºC and 75% relative humidity over a period of six months.
Long term shelf-stability testing was also performed on samples and stored at 25ºC and 60%
relative humidity over a period of twelve months. These shelf-life stability studies
demonstrated that HIDROX® is stable for 12 months when stored at 25ºC and 60% relative
humidity, when stored in well-closed containers and protected from light, moisture, and heat.
There are no changes in appearance, no substantial changes in the analysis for phenolics,
hydroxytyrosol, or ORAC value, all of which are key specifications for guaranteeing
antioxidant potential and, therefore, the intended technical function of HIDROX®. There were
no changes in total aerobic microbial count, total combined yeast and mold count and after
storage for 12 months in well-closed containers, protected from light, moisture, and heat, at a
temperature of 25ºC. Besides this study, we have tested production lots that are over three-year
old for microbial analysis and the results again show no changes. Based on these results, we
determined that a shelf life of two years is supported.

FDA Query: (4) In Table 3 you list the specifications for aqueous olive pulp extract liquid and
powder and the methods of analyses.

a) You list AOAC 925.09 as a method to assess the protein content, however this analytical
method is used to determine moisture content. Please provide the method used assess
protein content in aqueous olive pulp extract.

Page 2 of 8
 Response: a) Thank you for bringing this to our attention. The correct Protein Combustion (A)
Method Reference is: AOAC 990.03, AOAC 992.15

b) We also note that you cite “21 CFR-calc” as a reference for the method to assess the level
of carbohydrates in aqueous olive pulp extract. It is our understanding that this reflects
total carbohydrates (by calculation) and includes fiber. Please provide a more complete
description of where in 21 CFR the method is located.

Response: b) Thank you for pointing this. Your understanding about the method is correct.
The correct 21 CFR method is 21 CFR 101.9 (c)(6). A copy of this section is provided below.

(6) “Carbohydrate, total” or “Total carbohydrate”: A statement of the number of grams of

total carbohydrate in a serving expressed to the nearest gram, except that if a serving
contains less than 1 gram, the statement “Contains less than 1 gram” or “less than 1 gram”
may be used as an alternative, or if the serving contains less than 0.5 gram, the content may
be expressed as zero. Total carbohydrate content shall be calculated by subtraction of the
sum of the crude protein, total fat, moisture, and ash from the total weight of the food. This
calculation method is described in A. L. Merrill and B. K. Watt, “Energy Value of Foods -
Basis and Derivation,” USDA Handbook 74 (slightly revised 1973) pp. 2 and 3, which is
incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51 (the
availability of this incorporation by reference is given in paragraph (c)(1)(i)(A) of this
section).

c) You indicate a minimum value for total polyphenols and hydroxytyrosol but do not
indicate a maximum value. Please provide a range for these components in your
ingredient.

Response: c) The minimum and maximum values are as follows: For HIDROX® 12% the
polyphenol range is 12-16% (based on our experience over the years it is mostly 12-13%) and
the Hydroxytyrosol range is 3.5-4.5%. For HIDROX® 10X the polyphenol range is 7.5-8.5%
and the Hydroxytyrosol range is 3.0-4.0%.

FDA Query: (5) In Tables 4 and 5 you provide certificate of analyses data of aqueous olive pulp
extract liquid in comparison to aqueous olive extract pulp extract specifications.

a) Please clarify the composition of the ingredients by:

1. listing the major mineral components of ash

Response: For one of the HIDROX® 12% batch, Lot#12-180828-01 has been tested for
the following minerals. The available data from one lot is given in the below Table.

Page 3 of 8
 HIDROX 12% Lot #12-180828-001
Iron by ICP 0.0126%
Potassium by ICP 6.52%
Sodium by ICP 0.161%
Calcium by ICP 0.105%

2. indicating whether simple & polyphenols are expressed on a dry weight percent basis
or other basis.

Response: Please note the Simple & polyphenols are analyzed by the Folin-Ciocalteu
Assay and the results for HIDROX® 12% are expressed as the percentage mg of GAE
(Gallic Acid Equivalent) per g of dried sample. Sorry for not mentioning the units in the
Table.

b) In Table 4, for lot 12-140108-001, the batch analyses (0.133 mg/kg arsenic; 0.154 mg/kg
lead) do not meet stated specifications (both < 0.1 ppm (mg/kg) for these metals).
Please provide a statement that your ingredient will meet stated specifications. Further,
please revise the product specifications so that the specifications are reflective of your
lot analyses.

Response: Thank you very much for bringing this to our notice, we are sorry for the oversight.
We confirm that our ingredient (subject of the GRAS) will meet the specifications. To
support that our product meets the heavy metal specification, we are providing heavy metal
analysis from additional batches (please see Table below). This data from 5 different lots
of HIDROX® 12% suggest that the product meets established heavy metal specifications.
Please note that if the lot does not meet the established specifications, the lot will not be
marketed.

Lot Number
Parameter
Lot #12- Lot #12- Lot #12- Lot #12- Lot #12-
190926-000 190403-000 170623-000 180228-001 181206-001
Arsenic (As) 0.0295 0.037 0.042 0.034 0.033
(ppm)
Cadmium (Cd) <0.005 0.00519 <0.005 <0.010 <0.007
(ppm)
Lead (Pb) 0.0616 0.0583 0.033 0.037 0.032
(ppm)
Mercury (Hg) <0.005 <0.005 <0.007 <0.010 <0.007
(ppm)
Report Date 01/14/20 05/20/19 08/23/17 10/05/18 01/18/19
Eurofins

Page 4 of 8
FDA Query: (6) You provide an estimate of exposure of 1500 mg/p/d of the ingredient at the
90th percentile of intake. Please provide an estimate of mean consumption of this
ingredient.

Response: Sorry for not mentioning this in the GRAS dossier. The cumulative mean consumption
of hydrolyzed aqueous olive pulp extract from the proposed uses is estimated as 843 mg/p/d.

FDA Query: (7) Pages 22 (Table 8) and 28: For the summary of the acute toxicity study in rats,
the notifier states that “no adverse effects except soft or liquid feces” (Christian et al., 2004).
During the review of the original publication, FDA was unable to find the note regarding soft
or liquid feces in the acute study. FDA also notes that the publication by Christian et al.
(2004) states that “In rats, an acute oral NOAEL of 1000 mg/kg was established, based on
partial reductions in weight gains in both sexes at 5000 mg/kg, and reduced weight gains in
female rats at 1500 and 2000 mg/kg.” Therefore, please clarify what effects were seen in the
acute rat study and based on what effects the NOAEL was established.

Response: We are sorry for our confusion and the oversight. Based on the findings from the acute
oral toxicity study in rats, Christian et al. (2004) considered the acute oral NOAEL of
Hydrolyzed Aqueous Olive Pulp Extract as 1000 mg/kg bw (Christian et al., 2004). This was
determined as the NOAEL based on the observation that at higher doses of 1500 and 2000
mg/kg bw reduced body weight gains noted in female rats, while at the highest dose level of
5000 mg/kg bw, both males and females continued to gain weight, although at a reduced rate
as compared to control rats (Christian et al., 2004).

FDA Query: (8) On page 24 the notifier states “The results of this study suggest that the
resulting all user maximum intake of 25 mg/kg body weight (bw)/day from the proposed
uses of Aqueous Olive Pulp Extract (Hidrox®), the NOAEL is 80-fold lower.” This statement, as
written, does not make sense. Please concur whether you meant to state that the all user
maximum intake of 25 mg/kg bw/day resulting from the proposed uses of Aqueous Olive
Pulp Extract (Hidrox®) is 80-fold lower than the NOAEL obtained in the subchronic toxicity
study by Christian et al. (2004).

Response: We concur with the above statement and apologize for the confusing statement.

FDA Query: (9) On page 29 regarding the Bitler et al. (2007) study, the notifier states that
“Although the levels of hydroxytyrosol were not reported in the publication, given the

Page 5 of 8
affiliation of the authors of this study, the extract used in this study appears to be the
subject of this present GRAS assessment and the resulting intake of hydroxytyrosol appear
to be approximately 10 mg/person/day.”

a) FDA notes that seven of the nine authors of the publication by Bitler et al. (2007) are
affiliated with Arizona State University and two are affiliated with CreAgri, Inc. This GRAS
notice was submitted on behalf Oliphenol LLC. Please explain how the affiliation of the
authors of the Bitler et al. (2007) article makes the extract used in that study to “appear to
be” the same as the subject of this notice.

Response: a) Please note that Dr. Roberto Crea, currently CEO of Oliphenol LLC (notifier)
and is also the founder President and CEO of CreAgri Inc. The Bitler et al. (2007) study,
conducted at Arizona State University, used freeze-dried olive vegetation water that is
manufactured similarly to the subject of present GRAS (with some minor differences).
However, the levels of hydroxytyrosol were not determined or reported in the publication. The
study product was reported to contain at least 6% simple phenols and polyphenols. The subject
of this GRAS Notice, hydrolyzed aqueous olive pulp extract powder and liquid form contains
>12.0 and >7.5% polyphenols, and >3.5 and >3.0% hydroxytyrosol, respectively. Hence, we
made some assumptions to determine the hydroxytyrosol content of study product. If the Bitler
et al. (2007) study product contains 6% phenols and polyphenols, it is likely that it may contain
around 2.5% hydroxytyrosol and the resulting intake of hydroxytyrosol from a 400 mg/day dose
of the extract used in the study will be approximately 10 mg/person/day. Hence, we indicated
that the likely intake of hydroxytyrosol in the Bitler et al. (2007) study to be 10 mg/person/day.

b) Appear does not indicate certainty. Is or is not the test article in the Bitler et al. (2007) the
same as the subject of this notice?

Response: b) Based on the available information, the subject of the Bitler et al. (2007) study is
substantially similar to the subject of present GRAS.

c) We also note that in its GRAS notice the notifier identifies the test article used in this
study as “Hidrox”, the notified substance, but the article never refers to the test article as
such. Please state whether you concur.

Response: c) As the product of Bitler et al. (2007) study is manufactured similarly to the subject
of current GRAS, we used the term Hidrox®, although it is not mentioned in the publication.
At the time CreAgri marketed a range of products containing different levels of polyphenols
under the name Hidrox®. We concur that Hidrox is not mentioned in the article.

d) We also note that the participants received 400 mg/day of the test article. You state that
this corresponds to 10 mg hydroxytyrosol (HT)/day. Given that the specifications for the
notified substance state that the hydroxytyrosol content of the notified substance is >3.5%,

Page 6 of 8
 the test article in the Bitler et al. (2007) study should provide 14 mg hydroxytyrosol/day at a
minimum if it was the same as the notified substance. Please explain the discrepancy.

Response: d) As described above in “Response a)”, the hydroxytyrosol content was calculated
by making some assumptions.

FDA Query: (10) The notifier discusses a published 90-day study in which rats of both sexes
were fed 0, 5, 50, or 500 mg of HT/kg bw/day via gavage (Auñon-Calles et al., 2013) and
states that “the investigators proposed the dose of 500 mg/kg bw/day as the NOAEL.
FDA notes that at 500 mg/kg bw/day slightly but significantly lower body weight (14%) in
males and body weight gains in both sexes were reported despite of the fact that the mean
food consumption was comparable in all dose groups for both sexes. Statistically significant
higher relative kidney weights were observed in males and females as related to body
weight. Some other statistically significant differences in organ weights relative to body
weight were observed in animals from the 500 mg/kg bw/day group compared to controls.
Moreover, at the end of the recovery period, higher relative and absolute testes weights in
males and higher absolute and relative liver and kidney weights in females were observed
compared to the control group. No pathological changes were reported in these organs. The
authors of this study assigned the No Observed Adverse Effect Level (NOAEL) to 500 mg/kg
bw/day. On the basis of significantly lower body weight in males, body weight gains in both
sexes, and some other statistically significant differences in organ weights, the Food and
Drug Administration assigns the NOAEL to 50 mg/kg bw/day for this study. Please state
whether you concur with FDA’s assignment of 50 mg/kg bw/day as the NOAEL, if not, explain
why not.
Response: We concur with FDA’s assigned NOAEL of 50 mg/kg/bw/day.

FDA Query: (11) Pages 31-32: The notifier states that rats “were administered VOO” (i.e.,
aqueous virgin olive oil) “at levels of 0, 100, 300, and 1,000 mg/kg bw/day in the drinking
water” (page 31). At the end of the study summary, the notifier states “The investigators
concluded VOO extract containing 15% of hydroxytyrosol did not induce effects that can be
considered of toxicological relevance, and proposed a NOAEL dose as 1,000 mg/kg bw/day of
pure hydroxytyrosol.”
FDA notes that these statements are contradictory as if the top dose was 1,000 mg VOO
containing 15% HT, the top HT level would be 150 mg/kg bw/day and not 1,000. FDA’s
review of the original publication by Rodrigues-Lara et al. (2019) revealed that “The VOO
extract was dissolved and administered in the drinking water in order to minimize the
manipulation of the animals. After calculating the median daily amount of water intake,
dilutions of the extract were prepared in milli Q water to achieve final different doses of

Page 7 of 8
 hydroxytyrosol: 100 mg/kg (low dose), 300 mg/kg (intermediate dose), and 1000 mg/kg
(high dose).” Therefore, the animals were administered 0, 100, 300, or 1,000 mg HT/kg
bw/day and not VOO. Please state whether you concur.
Response: We concur with FDA. We apologize for not clarifying this. Apparently, the confusion
arose from the abstract of the publication that states, “The sub-chronic study included 60 rats
distributed in three groups (n = 20: 10 males and 10 females) receiving daily different three
doses of the VOO extract in the drinking water during 90 days: (1) 100 mg/kg, (2) 300 mg/kg,
and (3) 1000 mg/kg”

We trust that 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 your questions.

Best regards,

Madhu Soni

Page 8 of 8
Santos, Marissa

From: Madhu Soni <sonim@bellsouth.net>

Sent: Monday, October 11, 2021 4:03 PM
To: Santos, Marissa
Subject: RE: [EXTERNAL] RE: GRN 000978 - Question for the Notifier

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 Ms. Santos,

I have received the responses from the notifier (Oliphenol) of GRN 000978 for the below described three
queries. Please see below responses included next to the query:
1. Please confirm that all materials used in the manufacturing process of hydrolyzed aqueous olive pulp extract
(HAOPE) are used in accordance with U.S. regulations.
Response: We confirm that all materials used in the manufacturing of HAOPE are in accordance with U.S.
regulation.
2. On page 8 (Table 3), the specification for moisture in the liquid form of HAOPE is listed as 34% indicating that
there is no acceptable range for the moisture content. Please clarify whether there is a range of moisture
content that would be acceptable for the liquid form of HAOPE.
Response: We are sorry for not providing the range. Please note that the moisture content of liquid product
ranges from 30‐45%.
3. On page 8 (Table 3), please clarify the acronym “ELFA” under the Salmonella specification.
Response: We are sorry for the confusion, please note that Salmonella is routinely measured as per USP Chapter
62 method. By oversight ELFA (Enzyme‐Linked Fluorescent Assay) got inserted as sometime this method has also
been used.
Hope the above responses are satisfactory. Thank you for the opportunity to provide these clarifications. If
you have any questions, please let me know.
Best regards
Madhu

From: Santos, Marissa [mailto:Marissa.Santos@fda.hhs.gov]

Sent: Thursday, October 7, 2021 1:25 PM
To: Madhu Soni <sonim@bellsouth.net>
Subject: RE: [EXTERNAL] RE: GRN 000978 ‐ Question for the Notifier

Dear Dr. Soni,

During our review of GRAS Notice No. 000978, we noted additional questions that need to be addressed and are included
here:

1. Please confirm that all materials used in the manufacturing process of hydrolyzed aqueous olive pulp extract
(HAOPE) are used in accordance with U.S. regulations.

1
 2. On page 8 (Table 3), the specification for moisture in the liquid form of HAOPE is listed as 34% indicating that
there is no acceptable range for the moisture content. Please clarify whether there is a range of moisture
content that would be acceptable for the liquid form of HAOPE.
3. On page 8 (Table 3), please clarify the acronym “ELFA” under the Salmonella specification.

We respectfully request a response within 10 business days. If you are unable to complete the response within that time
frame, please contact me to discuss further options. Please do not include any confidential information in your
responses.

If you have any questions or need further clarification, please feel free to reach out to me.

Regards,
Marissa

Marissa Santos, M.S.

Regulatory Review Scientist
Division of Food Ingredients
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
U.S Food and Drug Administration
Tel: 240.402.8160
marissa.santos@fda.hhs.gov
The link ed image canno t be display ed. The file may hav e been mov ed, renamed, or deleted. Verify that the link points to the correct file and loc atio n .

From: Madhu Soni <sonim@bellsouth.net>

Sent: Monday, July 12, 2021 3:55 PM
To: Santos, Marissa <Marissa.Santos@fda.hhs.gov>
Subject: [EXTERNAL] RE: GRN 000978 ‐ Question for the Notifier

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 Ms. Santos,

Please find attached a pdf file providing a point-by-point response to the agency queries related to our GRAS
notification (GRN 000978) .

I hope the information and clarifications, along with some discussion in the response addresses the FDA queries.
If you have any questions or need additional explanation, please let me know.

Thank you for the opportunity to provide this explanation.

Best regards
Madhu
------------------------------------------
Madhu Soni, PhD, FACN, FATS
Soni & Associates Inc.
749 46th Square
Vero Beach, FL 32968, USA
Phone: +1-772-299-0746
Cell: +1-772-538-0104
2
www.soniassociatesnet

From: Santos, Marissa [mailto:Marissa.Santos@fda.hhs.gov]

Sent: Friday, June 25, 2021 1:55 PM
To: Madhu Soni <sonim@bellsouth.net>
Subject: GRN 000978 ‐ Question for the Notifier

Dear Dr. Soni,

During our review of GRAS Notice No. 000978, we noted several questions that need to be addressed and are attached
to this email.

We respectfully request a response wi thin 10 business days. If you are unable to

complete the response within that time frame, please contact me to discuss further options. Please do not include any
confidential information in your responses.

If you have any questions or need further clarification, please feel free to reach out to me.

Regards,
Marissa

Marissa Santos, M.S.

Regulatory Review Scientist
Division of Food Ingredients
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
U.S Food and Drug Administration
Tel: 240.402.8160
marissa.santos@fda.hhs.gov
The link ed image canno t be display ed. The file may hav e been mov ed, renamed, or deleted. Verify that the link points to the correct file and loc atio n .