Consultants 

December 13, 2017 

Nadine Bewry, Ph.D., MPH 

Consumer Safety Officer/Toxicology Reviewer 

U.S. Food and Drug Administration 

Office of Food Additive Safety 

Division of Biotechnology and GRAS Notice Review 

P: 240-402-1007 

Nadine.bewry@fda.hhs.gov 

859 Outer Road 

Orlando, Florida 32814 

p•407.802.1400 

.f•407.802-1405 

e•rmatulka@burdockgroup.com 

Re: Request to Cease to Evaluate GRN000716 

Dear Dr. Bewry, 

Two conference calls were conducted in November 2017 between FDA's Office of Food 

Additive Safety (OFAS) and Burdock Group (agent for Arla Foods Ingredients Group PIS, 

sponsor of GRN000716). The subject of both of these calls was the notification of the conclusion 

of GRAS status completed for the bovine whey-derived osteopontin-based ingredient Lacprodan 

OPN-10, an ingredient to be added to term infant formula and powdered beverages targeted for 

children 1-3 years of age. The GRAS notification was received by FDA on July 10, 2017 and 

was accepted for filing by FDA on August 3, 2017. FDA provided questions on October 3, 2017 

requesting clarification (generally) on methodology, safety factors, the levels of osteopontin in 

breast milk, breast milk osteopontin cleavage variations stated in the literature, and small but 

significant plasma threonine levels found in a clinical trial evaluating the ingredient in infant 

formula. None of these questions indicated significant toxicological concerns, and the notifier 

addressed these questions in a timely fashion (response provided by October 20, 2017). During 

the teleconferences it was requested by FDA that the notification be withdrawn. While an earlier 

in-person conference (February 24, 2015) with FDA did not indicate that FDA had safety 

concerns and the questions provided by FDA did not suggest significant concerns of the safety of 

Lacprodan OPN-10 under the intended conditions of use, during the recent conference calls it 

was indicated that there were inadequacies in the notification, not otherwise identified in your 

letter of April 16, 2015 or the request for additional clarification dated October 3, 2017. 

Although the extent or specific types of newly announced inadequacies was not made 

clear during the November conference calls, FDA indicated it would deliver a clarifying memo 

and/or memorialized minutes of the call to identify the Agency's newly discovered inadequacies 

with the notification; unfortunately, we still await this written information. We have great 

expectations that this written memorandum will be provided in the near future. Nonetheless, in 

December :1.3, 20:1.7 

fusing science and compliance 

13.ARLAOOJ.06 

CONFIDENTIAL 

Page:1.of2 

www.burdockgroup.com 
the spirit of cooperation with the Agency, we will comply with your request and ask that your 

office cease to evaluate GRAS notification 000716. 

December i3, 2017 

fusing science and compliance 

13.ARLAOOJ.06 

CONFIDENTIAL 

Sincerely, 

Ray A. Matulka, Ph.D. 

Director of Toxicology 

Burdock Group 

Page2of2 

www.burdockgroup.com 
From: Ray Matulka

To: Bewry, Nadine

Cc: Carrie Kennedy

Subject: RE: GRN 000716 (bovine whey-derived osteopontin (bOPN)): Meeting memorandum

Date: Thursday, December 21, 2017 12:10:34 PM

Attachments: image008.png

Dear Dr. Bewry,

We have reviewed the memorandum that you provided yesterday, and the client has

requested that a statement made within the memorandum be modified, as we do not feel that the

statement completely reflects the information that has been provided within the notification

documents.

 The statement that requires clarification is the following:

“There is no evidence provided in GRN 000716 or in the October 20, 2017 amendment to GRN

000716 that infants have been safety exposed to higher levels of bOPN from the intended use.”

We believe that the 6-month clinical trial conducted by Lonnerdal et al (2016) that was referenced in

GRN 0000716 provides growth (i.e., anthropometry), formula intake and adverse event evaluations,

consistent with evaluating safety of infants. This study indicates no serious adverse events when

infants consume bOPN (i.e., Lacprodan OPN-10, the new ingredient) at levels at the intended use

level.

We would appreciate it that the above-stated sentence be removed from the memorandum, as we

do not feel that it accurately reflects the scientific research that has been completed on OPN-10, and

the lack of concern from the pediatric community on the results provided in this peer-reviewed,

published (Journal of Pediatric Gastroenterology and Nutrition) clinical trial. 

Please contact me if you have any questions concerning this request.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

Burdock Group Consultants

Fusing Science & Compliance. Worldwide.

859 Outer Road

Orlando, FL 32814

P: (407) 802-1400 ext. 164

F: (407) 802-1405
______________________________________________________________________________________________________________________________ 

Memorandum of Meeting

Type Teleconference

Dates & 

Times

November 9, 2017, 11:00 a.m. – 12:00 p.m.

November 20, 2017, 11:00 a.m. – 11:30 a.m.

Location FDA, Center for Food Safety and Applied Nutrition, Office of Food Additive 

Safety, 4300 River Road, College Park, MD 20740

Subject: GRN 000716 (Bovine whey-derived osteopontin (bOPN)) for use as a source of 

protein in milk-based, non-exempt infant formulas for term infants and in powdered beverages 

at levels up to 138 mg/L as consumed.

Summary: This memorandum summarizes the discussion points of OFAS’ teleconference 

meetings with Burdock Group Consultants (agent) on November 9, 2017 and with Burdock 

Group Consultants and Arla Foods Ingredients Group PIS (AFI, notifier) on November 20, 

2017 regarding GRN000716. OFAS has outstanding regarding the evidence for general 

recognition that bOPN is safe for use in infant formulas. The overarching question is whether

the data provided by AFI are adequate to demonstrate general recognition of safety (reasonable 

certainty of no harm) by scientists with the appropriate expertise to evaluate the significance of 

bOPN activity at the intended consumption level by infants. OFAS considers this question to be 

important given the rapid development of the infant immune system, the poorly characterized 

modes of action for bOPN, the potential involvement of human OPN (hOPN) in infant immune 

maturation, lack of clarity regarding substantial equivalence of biological activities between 

bOPN and hOPN, and the large variation of the levels of hOPN in human milk.

On October 3, 2017, OFAS sent questions to AFI (see FDA Questions and Comments). On 

October 20, OFAS received AFI’s responses. The amendment included information on the 

intended use level and resulting exposure to bOPN in infants. The amendment also included 

information on the bioequivalence of hOPN and bOPN. After reviewing AFI’s responses, OFAS 

continues to question the general recognition of the safety of the intended use of bOPN in 

infant formulas. 

On November 9, 2017 OFAS held a teleconference meeting with AFI’s agents, Drs. Burdock and 

Matulka, to discuss the review team’s outstanding questions regarding the GRAS status of the 

intended use of bOPN. 
Participants: November 9, 2017 Meeting

Notifier: Arla Foods Ingredients Group PIS (AFI)’| Agent: Burdock Group Consultants (phone)

George A. Burdock, Ph.D. Owner, Burdock Group Consultants

Ray A. Matulka, Ph.D. Director of Toxicology, Burdock Group Consultants

FDA/CFSAN/ OFAS/DBGNR (HFS-255)

Nadine Bewry, Ph.D., MPH Consumer Safety Officer (CSO)

Jeremiah Fasano, Ph.D. Acting Supervisory CSO

Romina Shah, Ph.D. Chemist

Michael DiNovi, Ph.D. (phone) Supervisory Chemist

Kotaro Kaneko, Ph.D. Toxicologist

Ronald Chanderbhan, Ph.D. Supervisory Toxicologist

FDA/CFSAN/ ONFL/ IFMS (HFS-850) (phone)

Linda Tonucci, Ph.D. CSO

Suzanne Wolcoff, PhD. Senior Dietitian-Nutritionist

Carrie Assar, Ph.D. Lead Nutritionist

OFAS discussed three main lines of evidence contained in the notice and in the notifier’s 

response to one of OFAS’ questions, including the absence of evidence of toxicity in 

toxicological and clinical studies, the similarity of bOPN and hOPN, and existing infant 

exposure to both bOPN and hOPN. 

OFAS noted that:

o OPN has multiple modes of action, including immunomodulatory and pro-inflammatory 

effects. None of the published safety studies discussed in GRN 000716 evaluated 

potential adverse effects of bOPN at the intended use level related to these modes of 

action.

o The available evidence indicates that hOPN and bOPN are not substantially 

bioequivalent in humans from a physiological perspective. 

o The intended use level of bOPN in infant formula is higher than the basal level found in 

milk or milk-based formula. 

 Based on the literature, the levels of bOPN in cow’s milk and cow’s milk-derived 

infant formulas are 18 μg/ml and 5.3-13.0 μg/ml, respectively. However, levels of 

bOPN from intended use is ~138 μg/ml. 

 There is no evidence provided in GRN 000716 or in the October 20, 2017 

amendment to GRN 000716 that infants have historically been safely exposed to 

bOPN containing dairy products at levels as high as the intended use level.

OFAS considers it important that AFI provide evidence that the existing data and information

are adequate to demonstrate general recognition of safety (reasonable certainty of no harm) by 

scientists with the appropriate expertise to evaluate the significance of bOPN activity at the 

intended use level in infants. OFAS further noted that AFI’s GRAS Panel appears to lack
expertise in neonatal immunology, which is relevant to the intended population and the known 

modes of action of bOPN. OFAS explained that the importance of including such expertise 

would be to develop insight into the views of scientists in this area about the significance of the 

intended use of bOPN, including some basis for concluding that these views are broadly 

shared. OFAS informed AFI that simply adding a neonatal immunologist to their current GRAS 

panel would not be sufficient to provide evidence of general recognition. The introduction of an 

additional individual without reanalysis and interpretation of existing data or reference to 

current thinking in their field would not be sufficient to resolve our questions about general 

recognition. Furthermore, OFAS noted that other regulatory authorities, including EFSA, 

JECFA, FSANZ, and Health Canada, have not approved the use of bOPN in infant formula, 

indicating a lack of general recognition of bOPN’s safety in infant formulas. Given these 

outstanding questions regarding general recognition, OFAS advised AFI to request that OFAS 

cease to evaluate GRN 000716.

On November 15, 2017, Dr. Matulka requested a second teleconference meeting with OFAS

because AFI stated that they would like to address OFAS’ questions regarding the notice rather 

than request that OFAS cease to evaluate the notice. The purpose of the meeting was: (1) to

share the steps that AFI will take to address our questions, and (2) to gain clarification on some

of the discussion points from the November 9, 2017 teleconference meeting.

On November 20, 2017, OFAS held a second teleconference meeting with AFI and AFI’s agents.

Participants: November 20, 2017 Meeting

Notifier: Arla Foods Ingredients Group PIS (AFI)’| Agent: Burdock Group Consultants (Phone)

George A. Burdock, Ph.D. Owner, Burdock Group Consultants

Ray A. Matulka, Ph.D. Director of Toxicology, Burdock Group Consultants

Carrie Kennedy, PMP, RAC Project Leader, Burdock Group Consultants

Kal Ramanujam, Ph.D. Senior Scientific Advisor, Arla Foods

Anders Steen Jorgensen Head, Business Unit Pediatrics, Arla Foods

Anne Staudt Kvistgaard Senior Manager, Documentation Pediatrics, Arla Foods

FDA/CFSAN/ OFAS/DBGNR (HFS-255)

Nadine Bewry, Ph.D., MPH Consumer Safety Officer (CSO)

Shayla West-Barnette, Ph.D. Supervisory CSO

Jeremiah Fasano, Ph.D. Acting Supervisory CSO

Rachel Morissette, Ph.D. CSO

Kotaro Kaneko, Ph.D. (phone) Toxicologist

Supratim Choudhuri, Ph.D. Acting Supervisory Toxicologist

FDA/CFSAN/ ONFL/ IFMS (HFS-850) (Phone)

Carrie Assar, Ph.D. Lead Nutritionist

Andrea Lotze, M.D. Medical Officer

OFAS’ discussion points included some of those already covered during the November 9, 2017 

teleconference meeting, as well as the following discussion points:
o OFAS considers that it would not be possible for AFI to address the unresolved 

questions regarding general recognition of the safety of the intended use of bOPN in

the short term simply by locating or conducting additional studies.

o OFAS emphasized the office’s view that the unresolved questions rest on the issue of 

whether scientists trained to evaluate immune function and development in infants

would accept the existing evidence as adequate to show reasonable certainty of no 

harm.

o OFAS noted that there are several potential strategies to develop evidence for general

recognition of the safety of the intended use of bOPN, which would involve recruiting 

additional expertise and re-engaging with OFAS on the issues we identified.

o OFAS noted that any future consideration of the GRAS status of AFI’s intended use 

of bOPN by the office would include consulting FDA staff with appropriate training 

and expertise relevant to neonatal immune development. 

In conclusion, OFAS still has outstanding questions regarding the evidence for general 

recognition that bOPN is safe for use in infant formulas. OFAS recommends that AFI request 

that we cease to evaluate GRN 000716.

Attachment:

GRN 716 2017-10-03 Email_FDA Questions and Comments

______________________________

Nadine Bewry, Ph.D., MPH

Nadine N. Bewry -S

Digitally signed by Nadine N. Bewry -S 

DN: c=US, o=U.S. Government, ou=HHS, ou=FDA, 

ou=People, 0.9.2342.19200300.100.1.1=0014360008, 

cn=Nadine N. Bewry -S 

Date: 2018.01.25 13:49:49 -05'00'
R/D:HFS-255:NNBewry: 11/20/2017

Edit/Comments/Init: HFS-255: RShah:12/5/2017

Edit/Comments/Init: HFS-255: KKaneko:12/4/2017, 12/7/2017, 12/22/2017

Edit/Comments/Init: HFS-255: JFasano:12/4/2017, 12/7/2017

Edit/Commens:HFS-255: SWestBarnette:12/19/2017, 12/20/2017

Init:HFS-255: SWestBarnette:12/20/2017

F/T: HFS-255:NNBewry: 12/20/2017
Ray A. Matulka, Ph.D.

Director of Toxicology

Burdock Group Consultants

859 Outer Road

Orlando, FL 32814

Re: GRAS Notice No. GRN 00716

Dear Dr. Matulka:

This letter corrects our letter in response to GRN 000716 dated January 30, 2018. The

purpose of this revised letter is to correct the date on which you asked FDA to cease to

evaluate GRN 000716.

The Food and Drug Administration (FDA, we) is granting Arla Foods Ingredients Group 

PIS (AFI)’s request to cease our evaluation of GRN 000716, which we filed on August 3, 

2017. We received your request on December 13, 2017.

The subject of the notice is bovine whey-derived osteopontin (bOPN) for use as an 

ingredient in milk-based, non-exempt infant formulas for term infants and in powdered 

beverages at levels up to 138 mg/L as consumed. The notice informs us of AFI’s view 

that this use of bOPN is GRAS through scientific procedures.

On October 3, 2017, we sent questions to AFI. After reviewing AFI’s responses, we have 

questions regarding the intended use of bOPN in infant formulas. In a telephone 

conversation with Burdock Group Consultants (Burdock Group) on November 9, 2017, 

and in a telephone conversation with Burdock Group and AFI on November 20, 2017,

we discussed our outstanding questions. We explained that the amendment we received 

from AFI on October 20, 2017, did not fully address our questions. The amendment 

included information on the intended use level and resulting exposure to bOPN in 

infants. The amendment also included information on the bioequivalence of human 

OPN and bOPN. We also discussed the opportunity for AFI to ask us to cease our 

evaluation of GRN 000716.
Page 2 – Dr. Matulka

In accordance with 21 CFR 170.275(b)(3), the text of this letter responding to GRN 

000716 is accessible to the public at www.fda.gov/grasnoticeinventory.

Sincerely,

Susan J. Carlson, Ph.D.

Director

Division of Biotechnology 

and GRAS Notice Review

Office of Food Additive Safety

Center for Food Safety 

and Applied Nutrition
Page 3 – Dr. Matulka

cc: GRN 000716

Electronic mail cc: 

HFS-200 (DKeefe, MAdams)

HFS-255 (SCarlson, RFChanderbhan, MDiNovi, RIMerker, SWestBarnette, PMGaynor, 

RShah, KKaneko, RBonnette, LShepherd))

HFS-850 (ALotze, CAssar) 

R/D:HFS-255:NNBewry:12/14/2017

Edit/Comment:HFS-255:SWestBarnette:12/19/17, 12/20/17

Init:HFS-255:SWestBarnette:12/20/17, 02/01/2018

Edit/Comment:PMGaynor:01/11/2018, 1/30/2018Comment:HFS255:KKaneko:12/22/2017

Comment:HFS-255:JFasano:01/26/2018

Edit:HFS-255:NNBewry: 12/15/2017, 1/12/2018, 1/22/2018, 1/24/2017, 1/29/2017, 

1/30/2018, 1/31/2018, 02/01/2018

Discussion and edits:HFS-255:SCarlson,SWestBarnette,PGaynor:01/30/2018

Init:HFS-255: SCarlson:01/30/2018, 01/31/2018

F/T:HFS-255: NNBewry:02/01/2018

NAME ELECTRONIC SIGN-OFF ACTING?

Nadine Bewry

Consumer Safety Officer

Susan Carlson

Director, Division of Biotechnology & GRAS 

Notice Review

Nadine N. 

Bewry -S

Digitally signed by Nadine N. 

Bewry -S 

DN: c=US, o=U.S. Government, 

ou=HHS, ou=FDA, ou=People, 

0.9.2342.19200300.100.1.1=0014

360008, cn=Nadine N. Bewry -S 

Date: 2018.02.01 10:40:01 -05'00'

Susan J. 

Carlson -

S

Digitally signed by Susan J. 

Carlson -S 

DN: c=US, o=U.S. 

Government, ou=HHS, 

ou=FDA, ou=People, 

0.9.2342.19200300.100.1.1=20

00419015, cn=Susan J. 

Carlson -S 

Date: 2018.02.01 12:01:51 

-05'00'
Ray A. Matulka, Ph.D. 

Director of Toxicology 

Burdock Group Consultants 

859 Outer Road 

Orlando, FL 32814 

Re: GRAS Notice No. GRN 000716 

Dear Dr. Matulka:

Enclosed is a revised copy of the response letter for GRN 000716. The original letter for 

this GRAS notice was signed on January 30, 2018. In an electronic mail message dated 

January 30, 2018, you informed us that Arla Foods Ingredients Group PIS (AFI) 

requested that FDA cease to evaluate GRN 000716 on December 13, 2017, not December 

13, 2016.

In response to your electronic mail message, we have revised the response letter to 

reflect the correct date of the cease-to-evaluate request. We regret any inconvenience 

that our error may have caused. If you have any questions, please contact me by 

electronic mail at Nadine.Bewry@fda.hhs.gov or by telephone at 240-402-1007. 

 Sincerely, 

 Nadine Bewry, Ph.D., MPH 

 Division of Biotechnology 

 and GRAS Notice Review 

 Center for Food Safety 

 and Applied Nutrition 

Nadine N. 

Bewry -S

Digitally signed by Nadine N. Bewry -S 

DN: c=US, o=U.S. Government, 

ou=HHS, ou=FDA, ou=People, 

0.9.2342.19200300.100.1.1=001436000

8, cn=Nadine N. Bewry -S 

Date: 2018.02.01 16:40:11 -05'00'
cc: GRN 000716

R/D:HFS-255:NNBewry:01/31/2018

Edit/Comment:HFS-255:SWestBarnette: 2/1/18 

Init:HFS-255:SWestBarnette: 2/1/18 
Ray A. Matulka, Ph.D. 

Director of Toxicology 

Burdock Group Consultants 

859 Outer Road 

Orlando, FL 32814 

Re: GRAS Notice No. GRN 000716 

Dear Dr. Matulka:

Enclosed is a revised copy of the response letter for GRN 000716. The original letter for 

this GRAS notice was signed on January 30, 2018. In an electronic mail message dated 

January 30, 2018, you informed us that Arla Foods Ingredients Group PIS (AFI) 

requested that FDA cease to evaluate GRN 000716 on December 13, 2017, not December 

13, 2016.

In response to your electronic mail message, we have revised the response letter to 

reflect the correct date of the cease-to-evaluate request. We regret any inconvenience 

that our error may have caused. If you have any questions, please contact me by 

electronic mail at Nadine.Bewry@fda.hhs.gov or by telephone at 240-402-1007. 

 Sincerely, 

 Nadine Bewry, Ph.D., MPH 

 Division of Biotechnology 

 and GRAS Notice Review 

 Center for Food Safety 

 and Applied Nutrition 

Nadine N. 

Bewry -S

Digitally signed by Nadine N. Bewry -S 

DN: c=US, o=U.S. Government, ou=HHS, 

ou=FDA, ou=People, 

0.9.2342.19200300.100.1.1=0014360008

, cn=Nadine N. Bewry -S 

Date: 2018.02.01 16:54:30 -05'00'
From: Bewry, Nadine

To: Ray Matulka (RMatulka@burdockgroup.com)

Subject: GRN 000716 (bovine OPN) Corrected Response Letter

Date: Thursday, February 1, 2018 5:02:20 PM

Attachments: GRN 716 Correction Letter.pdf

GRN 716 2018-01-31 Corrected Cease to Evaluate Response Letter.pdf

image001.png

Dear Dr. Matulka,

FDA’s corrected “cease-to-evaluate” response letter to GRN 000716 was signed today by our

Division Director, Dr. Susan Carlson. I have also attached a letter that describes the

correction. Both letters are attached.

To address your question, the information that will be available publicly on our website are

the following:

1. GRN 000716,

2. The 10-20-2017 amendment to GRN 000716 (response to FDA’s questions and

comments), and

3. FDA’s cease-to-evaluate response letters (the original and the corrected versions).

Please let me know if you have any questions.

Best regards,

Nadine

Nadine Bewry, PhD, MPH

Consumer Safety Officer | Toxicology Reviewer

Division of Biotechnology and GRAS Notice Review

Office of Food Additive Safety

Center for Food Safety and Applied Nutrition

5001 Campus Drive, HFS-255

College Park, MD 20740
From: Ray Matulka

To: Carlson, Susan

Cc: Bewry, Nadine; Carrie Kennedy

Subject: RE: Burdock checking on scientific memorandum for OPN

Date: Monday, June 18, 2018 5:12:01 PM

Attachments: image001.png

image032.png

image038.png

image039.png

image040.png

image041.png

Arla-Letter to FDA-actions forward-signed-2018JUN18.pdf

Dear Dr. Carlson,

 First, I wanted to thank you for your previous update on the status of the bovine

osteopontin memoranda that are being prepared as part of the record for GRN 000716, if I did not

previously.

 However, Arla is anxious to move forward to address concerns within your group on the

safety of this bovine osteopontin ingredient. Therefore, Arla requested that I provide your group the

attached letter indicating the planned actions to address the concerns stated in the previous

meetings with your group. I will follow this email with a certified mailing of the original letter, for

your records.

 Please let me know if you have any questions. 

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

PH: 407-802-1400, x 164

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Carlson, Susan [mailto:Susan.Carlson@fda.hhs.gov] 

Sent: Thursday, April 19, 2018 4:57 PM

To: Ray Matulka

Cc: Bewry, Nadine; Carrie Kennedy

Subject: RE: Burdock checking on scientific memorandum for OPN

Dear Dr. Matulka,

I want to give you an update on the status of the bovine osteopontin memoranda that we are

preparing as part of the record for GRN 000716.

We are still working on the memoranda. I can assure you that these documents are at the forefront

of our work. Indeed, we have been having numerous internal conversations with our colleagues
about the issues raised by the proposed use of this substance. We are working to further refine our

thoughts and written record in a manner that will be useful to us and your client. I will share with

you that there are drafts that are circulating for comment and there will need to be some further

work on the documents by the team.

As you are well aware, the GRAS concept offers much in the way of regulatory flexibility; where we

must take a pause is when we are asked to assess a substance like osteopontin in that it lacks a

counterpart in our program. We believe that this substance warrants our best thinking and we hope

that you and your client can be patient for a bit longer. I am sure that you are anxious for a date. I’ve

learned over the years that there are too many variables in our work to accurately project

completion dates. However, I invite you to continue to check in with us on a regular basis because it

does help to keep the pressure on us. We don’t like to leave projects undone—there isn’t any

satisfaction in that for anyone!

I would also like to take this opportunity to thank you for your continued participation in our GRAS

Notification Program. We would not be able to do the work that we do without the dedicated efforts

of our stakeholders like you.

Sincerely,

Susan J. Carlson, Ph.D.

Division Director 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety, Division of Biotechnology and GRAS Notice Review

U.S. Food and Drug Administration

Tel: 240-402-1253

Susan.Carlson@fda.hhs.gov

From: Bewry, Nadine 

Sent: Wednesday, April 18, 2018 5:13 PM

To: Carlson, Susan <Susan.Carlson@fda.hhs.gov>

Subject: RE: Burdock checking on scientific memorandum for OPN

From: Ray Matulka [mailto:RMatulka@burdockgroup.com] 

Sent: Tuesday, April 17, 2018 9:43 AM

To: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>

Cc: Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)
Dear Dr. Bewry,

I was wondering if there has been any indication on the possible timing of the release of the

scientific memorandum for the Arla GRN (see below)?

Any information you can provide would be appreciated.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Bewry, Nadine [mailto:Nadine.Bewry@fda.hhs.gov] 

Sent: Tuesday, April 3, 2018 11:18 AM

To: Ray Matulka

Cc: Carrie Kennedy

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Good morning Dr. Matulka,

Thank you for checking-in on the memoranda and for your patience. I understand the

notifier’s desire to move forward. Please know that I share your correspondences with

the review team and I check on the status of both memoranda regularly.

The reference to a “few days” that you mentioned in your email below was pertaining

to the completion of the draft scientific memorandum. At the time of our meeting,

both memoranda were in different stages of development.

Currently, the scientific and policy memoranda are undergoing the office’s internal

review and clearance process and the team is working diligently to complete that

process.

Best regards,

Nadine Bewry, PhD, MPH

Consumer Safety Officer | Toxicology Reviewer

From: Ray Matulka [mailto:RMatulka@burdockgroup.com] 

Sent: Monday, April 2, 2018 1:48 PM
To: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>

Cc: Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Bewry,

 I am inquiring concerning the completion of the scientific and policy memorandum for GRN

000716, as previously discussed in the FDA meeting held on March 1, 2018: have those memoranda

been finalized, such that I can request a FOIA for them?

 Please let me know at your earliest convenience.

The client is VERY anxious to move forward with addressing the questions posed by your

team, but was indicated during the above-mentioned meeting to utilize the scientific memo as a

guide to addressing these questions, and we do not want to deviate from what is expected by your

group by moving forward without this information.

Several people at the meeting indicated that the memo was only a “few days” from

acceptance... it has now been a month since the meeting.

 Is there another person that I should be contacting to move this process forward, in

addition to yourself?

Any help you can provide is appreciated. 

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Bewry, Nadine [mailto:Nadine.Bewry@fda.hhs.gov] 

Sent: Monday, March 26, 2018 1:14 PM

To: Ray Matulka

Cc: Carrie Kennedy

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Matulka,

The documents are not available at this time. Thanks for following up.

Best regards,

Nadine Bewry, PhD, MPH
Consumer Safety Officer | Toxicology Reviewer

From: Ray Matulka [mailto:RMatulka@burdockgroup.com] 

Sent: Monday, March 26, 2018 11:28 AM

To: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>

Cc: Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Bewry,

 I am inquiring concerning the completion of the scientific and policy memorandum for GRN

000716, as previously discussed in the FDA meeting held on March 1, 2018: have those memoranda

been finalized, such that I can request a FOIA for them?

 Please let me know at your earliest convenience.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Bewry, Nadine [mailto:Nadine.Bewry@fda.hhs.gov] 

Sent: Wednesday, March 21, 2018 4:26 PM

To: Ray Matulka

Subject: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Matulka,

Attached, please find the meeting memorandum. I also included a copy of the draft

guidance document, Best Practices for Convening a GRAS Panel: Guidance for

Industry.

Please let me know if you have any questions.

Best regards,

Nadine Bewry, PhD, MPH

Consumer Safety Officer | Toxicology Reviewer

Office of Food Additive Safety

Center for Food Safety and Applied Nutrition


From: Carlson, Susan

To: Morissette, Rachel

Subject: FW: repeat...FW: Burdock checking on scientific memorandum for OPN

Date: Monday, March 04, 2019 11:43:18 AM

Attachments: image001.png

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From: Ray Matulka <RMatulka@burdockgroup.com>

Sent: Thursday, June 21, 2018 5:04 PM

To: Carlson, Susan <Susan.Carlson@fda.hhs.gov>

Cc: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>; Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: repeat...FW: Burdock checking on scientific memorandum for OPN

Dear Dr. Carlson,

                Thank you for sending this email (and acknowledging receipt of the email). As I am

somewhat confused on the “many safety questions” concerning the safety of the bovine

osteopontin (previous statements both on conference calls and in person focused on meeting the

“general recognition” standard), I look forward to the scientific and policy memos.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

PH: 407-802-1400, x 164

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Carlson, Susan [mailto:Susan.Carlson@fda.hhs.gov]

Sent: Thursday, June 21, 2018 4:53 PM

To: Ray Matulka

Cc: Bewry, Nadine; Carrie Kennedy

Subject: repeat...FW: Burdock checking on scientific memorandum for OPN

Dr. Matulka--

I’m sending this a second time as I see that Outlook has automatically inserted quotes in your email

address.

Please acknowledge that you have received this email.
Thank you,

Susan

From: Carlson, Susan

Sent: Thursday, June 21, 2018 4:46 PM

To: 'Ray Matulka' <RMatulka@burdockgroup.com>

Cc: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>; Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Burdock checking on scientific memorandum for OPN

Dear Dr. Matulka,

Thank you for your message. I would also like to thank you and Arla for your continued patience

regarding the finalizing of our memoranda.

I am happy to tell you that the memoranda have undergone their final review and should be

available very soon (days). I will let Dr. Bewry inform you when the finalized memoranda are

available for request.

I would also like to briefly respond to the proposal from Arla. I would advise that Arla review our

memoranda before hiring experts to draft a white paper. I can certainly understand that they are

wanting to move along. I realize that we discussed general recognition with you and Arla at length,

however, I would caution that after careful review of the literature and further discussion with our

colleagues (including FDA scientists outside of CFSAN), we view the science surrounding osteopontin

as unsettled. We have identified many safety questions that we elaborate on in our memoranda.

These questions do not have obvious answers and will need considerable effort to resolve.

Therefore, we do not believe that Arla’s proposal to engage two experts to write a white paper will

be sufficient. At this point in time, it is unclear how the use of osteopontin in infant formula would

be GRAS.

We are happy to discuss our thoughts further.

Regards,

Susan J. Carlson, Ph.D.

Division Director 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety, Division of Biotechnology and GRAS Notice Review

U.S. Food and Drug Administration

Tel: 240-402-1253

Susan.Carlson@fda.hhs.gov
From: Ray Matulka [mailto:RMatulka@burdockgroup.com]

Sent: Monday, June 18, 2018 5:11 PM

To: Carlson, Susan <Susan.Carlson@fda.hhs.gov>

Cc: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>; Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Burdock checking on scientific memorandum for OPN

Dear Dr. Carlson,

                First, I wanted to thank you for your previous update on the status of the bovine

osteopontin memoranda that are being prepared as part of the record for GRN 000716, if I did not

previously.

                However, Arla is anxious to move forward to address concerns within your group on the

safety of this bovine osteopontin ingredient. Therefore, Arla requested that I provide your group the

attached letter indicating the planned actions to address the concerns stated in the previous

meetings with your group. I will follow this email with a certified mailing of the original letter, for

your records.

                Please let me know if you have any questions.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

PH: 407-802-1400, x 164

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Carlson, Susan [mailto:Susan.Carlson@fda.hhs.gov]

Sent: Thursday, April 19, 2018 4:57 PM

To: Ray Matulka

Cc: Bewry, Nadine; Carrie Kennedy

Subject: RE: Burdock checking on scientific memorandum for OPN

Dear Dr. Matulka,

I want to give you an update on the status of the bovine osteopontin memoranda that we are

preparing as part of the record for GRN 000716.

We are still working on the memoranda. I can assure you that these documents are at the forefront

of our work. Indeed, we have been having numerous internal conversations with our colleagues

about the issues raised by the proposed use of this substance. We are working to further refine our

thoughts and written record in a manner that will be useful to us and your client. I will share with

you that there are drafts that are circulating for comment and there will need to be some further

work on the documents by the team.
As you are well aware, the GRAS concept offers much in the way of regulatory flexibility; where we

must take a pause is when we are asked to assess a substance like osteopontin in that it lacks a

counterpart in our program. We believe that this substance warrants our best thinking and we hope

that you and your client can be patient for a bit longer. I am sure that you are anxious for a date. I’ve

learned over the years that there are too many variables in our work to accurately project

completion dates. However, I invite you to continue to check in with us on a regular basis because it

does help to keep the pressure on us. We don’t like to leave projects undone—there isn’t any

satisfaction in that for anyone!

I would also like to take this opportunity to thank you for your continued participation in our GRAS

Notification Program. We would not be able to do the work that we do without the dedicated efforts

of our stakeholders like you.

Sincerely,

Susan J. Carlson, Ph.D.

Division Director 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety, Division of Biotechnology and GRAS Notice Review

U.S. Food and Drug Administration

Tel: 240-402-1253

Susan.Carlson@fda.hhs.gov

From: Bewry, Nadine

Sent: Wednesday, April 18, 2018 5:13 PM

To: Carlson, Susan <Susan.Carlson@fda.hhs.gov>

Subject: RE: Burdock checking on scientific memorandum for OPN

From: Ray Matulka [mailto:RMatulka@burdockgroup.com]

Sent: Tuesday, April 17, 2018 9:43 AM

To: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>

Cc: Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Bewry,

I was wondering if there has been any indication on the possible timing of the release of the

scientific memorandum for the Arla GRN (see below)?
Any information you can provide would be appreciated.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Bewry, Nadine [mailto:Nadine.Bewry@fda.hhs.gov]

Sent: Tuesday, April 3, 2018 11:18 AM

To: Ray Matulka

Cc: Carrie Kennedy

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Good morning Dr. Matulka,

Thank you for checking-in on the memoranda and for your patience. I understand the

notifier’s desire to move forward. Please know that I share your correspondences with

the review team and I check on the status of both memoranda regularly.

The reference to a “few days” that you mentioned in your email below was pertaining

to the completion of the draft scientific memorandum. At the time of our meeting,

both memoranda were in different stages of development.

Currently, the scientific and policy memoranda are undergoing the office’s internal

review and clearance process and the team is working diligently to complete that

process.

Best regards,

Nadine Bewry, PhD, MPH

Consumer Safety Officer | Toxicology Reviewer

From: Ray Matulka [mailto:RMatulka@burdockgroup.com]

Sent: Monday, April 2, 2018 1:48 PM

To: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>

Cc: Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)
Dear Dr. Bewry,

                I am inquiring concerning the completion of the scientific and policy memorandum for GRN

000716, as previously discussed in the FDA meeting held on March 1, 2018: have those memoranda

been finalized, such that I can request a FOIA for them?

                Please let me know at your earliest convenience.

The client is VERY anxious to move forward with addressing the questions posed by your

team, but was indicated during the above-mentioned meeting to utilize the scientific memo as a

guide to addressing these questions, and we do not want to deviate from what is expected by your

group by moving forward without this information.

Several people at the meeting indicated that the memo was only a “few days” from

acceptance... it has now been a month since the meeting.

                Is there another person that I should be contacting to move this process forward, in

addition to yourself?

Any help you can provide is appreciated.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Bewry, Nadine [mailto:Nadine.Bewry@fda.hhs.gov]

Sent: Monday, March 26, 2018 1:14 PM

To: Ray Matulka

Cc: Carrie Kennedy

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Matulka,

The documents are not available at this time. Thanks for following up.

Best regards,

Nadine Bewry, PhD, MPH

Consumer Safety Officer | Toxicology Reviewer

From: Ray Matulka [mailto:RMatulka@burdockgroup.com]
Sent: Monday, March 26, 2018 11:28 AM

To: Bewry, Nadine <Nadine.Bewry@fda.hhs.gov>

Cc: Carrie Kennedy <CKennedy@burdockgroup.com>

Subject: RE: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Bewry,

                I am inquiring concerning the completion of the scientific and policy memorandum for GRN

000716, as previously discussed in the FDA meeting held on March 1, 2018: have those memoranda

been finalized, such that I can request a FOIA for them?

                Please let me know at your earliest convenience.

Sincerely,

Ray A. Matulka, Ph.D.

Director of Toxicology

This e-mail message is intended for the exclusive use of the recipient(s) named above. It may contain information that is protected, privileged, or confidential and

should not be disseminated, distributed or copied to anyone not authorized to receive this information. If you think you have received this e-mail message in error,

please e-mail the sender immediately at rmatulka@burdockgroup.com . 

From: Bewry, Nadine [mailto:Nadine.Bewry@fda.hhs.gov]

Sent: Wednesday, March 21, 2018 4:26 PM

To: Ray Matulka

Subject: Memorandum of Meeting on 03/01/2018: bovine osteopontin (bOPN)

Dear Dr. Matulka,

Attached, please find the meeting memorandum. I also included a copy of the draft

guidance document, Best Practices for Convening a GRAS Panel: Guidance for

Industry.

Please let me know if you have any questions.

Best regards,

Nadine Bewry, PhD, MPH

Consumer Safety Officer | Toxicology Reviewer

Office of Food Additive Safety

Center for Food Safety and Applied Nutrition
______________________________________________________________________________________________________________________________ 

Memorandum 

Date June 29, 2018 

From Kotaro J. Kaneko (HFS-255) 

Through Romina Shah (HFS-255) ________________________ 

Nadine Bewry (HFS-255) ________________________ 

Subject GRN 000716, scientific memorandum 

To Administrative File, GRN 000716 (bovine osteopontin) 

Keywords: osteopontin (OPN), early T-lymphocyte activation-1 (Eta-1), secreted 

phosphoprotein 1 (SSP1), 44kDa bone phosphoprotein, sialoprotein 1, uropontin, 

infant formula, immune development, developmental immunotoxicity, 

immun0modulatory, bioactive 

GRAS Notice GRN No. 000716 (GRN 716) informs the Food and Drug Administration (FDA, 

we) of Arla Foods Ingredients Group P/S’s (the notifier) view that use of bovine osteopontin 

(bOPN) in nonexempt formulas for term infants is generally recognized as safe at 125 mg/L. 

According to the notifier, formulations of cow’s milk-based infant formula (IF) already contain 

5 to 13 mg bOPN/L, thus the final bOPN concentration from added and endogenous bOPN 

would be ~138 mg/L. 

This memorandum discusses unresolved questions about certain properties of partially 

purified bOPN in the context of its intended use which were raised during the scientific review 

of GRN 000716. 

Introduction: 

Osteopontin protein (OPN), encoded by the osteopontin/SSP11 gene, is a N-glycosylated 

phosphoprotein with wide ranging biological activities that include biomineralization, bone 

and tissue remodeling, and immunomodulation (Clemente et al., 2016; Kahles et al., 2014; 

Rittling and Singh, 2015; Wang and Denhardt, 2008). For this reason, OPN has been referred 

as “secreted phosphoprotein 1 (SSP 1),” “44kDa bone phosphoprotein,” “sialoprotein 1,” 

 

1 NCBI Gene Name for osteopontin is SSP1 

Nadine N. Bewry -S Digitally signed by Nadine N. Bewry -S 

DN: c=US, o=U.S. Government, ou=HHS, ou=FDA, 

ou=People, 0.9.2342.19200300.100.1.1=0014360008, 

cn=Nadine N. Bewry -S 

Date: 2018.07.02 10:13:45 -04'00'

Romina 

Shah -S

Digitally signed by Romina Shah -S 

DN: c=US, o=U.S. Government, ou=HHS, 

ou=FDA, ou=People, cn=Romina Shah -

S, 

0.9.2342.19200300.100.1.1=2000418924 

Date: 2018.07.10 11:55:28 -04'00'
Page 2 - Administrative File, GRN 000716 

“uropontin,” and “early T-lymphocyte activation-1 (Eta-1).” OPN is expressed in vivo as 

secreted/extracellular and intracellular forms. Thus, OPN is found in many biological fluids 

and expressed in multiple tissues and multiple cell types including osteoblasts, osteoclasts, 

endothelial and epithelial cells, as well as in most cells of the immune system. OPN has 

multiple binding sites for Ca+2 and heparin, as well as to different subsets of integrins, in which 

specificity and affinity are highly dependent on splicing, post-translational modification, and 

proteolytic cleavage (Christensen and Sorensen, 2014; Clemente et al., 2016; Lund et al., 

2009). Therefore, both the primary amino acid sequence and numerous post-translational 

modifications likely contribute to the observed pleiotropic effects of OPN. As with many milk 

proteins, the full range of biological functions of OPN in humans, and in particular in infants, 

are not yet fully understood. 

Since OPN’s initial discovery in 1985, extensive research2 has gradually advanced our 

understanding of the molecular mechanisms of action as well as its functional roles in normal 

development and physiology and in pathophysiological states (Clemente et al., 2016; Denhardt 

et al., 2001; Iida et al., 2017; Kahles et al., 2014; Rittling and Singh, 2015; Wang and Denhardt, 

2008). Although OPN has been linked to a wide variety of biological functions, this scientific 

memo focuses on its potential effects on the developing immune system as this would be a 

safety endpoint of concern for infants. OPN has been reported to have multiple effects on the 

immune system in adult humans and in animal models [Fig. 1; (Denhardt et al., 2001; Iida et 

al., 2017; Kahles et al., 2014; Rittling and Singh, 2015)]. Induction of cellular and humoral 

immune responses is dependent on up-regulation of two sets of cytokines synthesized by their 

respective T helper cell subclasses (Th1 and Th2). OPN/Eta-1 has a profound effect on the 

activation of T lymphocytes by directing naïve CD4 T cells towards Th1 commitment and 

differentiation; this in turn results in up-regulating expression of Th1 cytokines, including IL12 and IFN-γ, leading to polarization of Th1 response relative to Th2 response (Lund et al., 

2009; Renkl et al., 2005; Shinohara et al., 2005). In addition, OPN has been reported to have 

effects on a number of other immune cell types including macrophages, dendritic cells, and 

neutrophils (Kahles et al., 2014; Lund et al., 2009; Wang and Denhardt, 2008). Thus OPN 

appears to have one or more roles as an immunomodulatory bioactive molecule by influencing 

the responses of numerous immune cell components to various stimuli, not least of which may 

be to promote the appropriate balance of the activities between the Th1/Th23 arms of the 

developing immune system. Furthermore, OPN’s anti-apoptotic properties in activated T cells 

underline the potential causal relationship between OPN and pro-inflammatory diseases such 

as multiple sclerosis and diabetes (Clemente et al., 2016; Hur et al., 2007; Kahles et al., 2014; 

Ma et al., 2014; Rittling and Singh, 2015; Wang and Denhardt, 2008). 

The perinatal immune system is significantly different from that of an older child or an adult. It 

undergoes profound changes and maturation after birth (Basha et al., 2014; Dowling and Levy, 

2014; Zhang et al., 2017). Birth to one year of age is considered a critical window of 

immunological maturation for infants (DeWitt et al., 2012a, b; Simon et al., 2015). As 

discussed in Neal-Kleuver et al. (2014), “...infant CMI (cell-mediated immunity) is polarized 

relative to the adult, and can predispose infants to specific immune responses and possible 

 

2 PubMed search using the terms “osteopontin” and “human” under default settings identified 5374 results (as of 

6/20/2018). 

3 OPN has also been associated with influencing the activity of another subset of T helper cells (Th17) distinct from 

Th1/Th2, which is also associated with pro-inflammatory autoimmune disorders (Du et al., 2017; Kourepini et 

al., 2014; Santamaria and Corral, 2013). 
Page 3 - Administrative File, GRN 000716 

adverse outcomes.” This awareness of possible adverse outcomes and specific vulnerabilities in 

infants related to CMI is important when interpreting the significance of clinical data and study 

results. This includes whether the study design, endpoints, and statistical power were sufficient 

to address these issues. For example, the newborn infant immune system typically shows 

strong Th2 but attenuated Th1 response (Adkins et al., 2004; Neal-Kluever et al., 2014; Simon 

et al., 2015). This Th2-Th1 balance shifts with growth and maturity such that in young children 

it is less skewed towards Th2. 

 

In light of OPN being a known promoter and polarizer of the Th1 arm of the adaptive immune 

system, one hypothesis consistent with the available information is that a particular level of 

hOPN in breastmilk may be associated with the maintenance of a genetically and 

environmentally appropriate Th2/Th1 balance for an individual infant. Variability of hOPN 

levels may thus reflect the health and/or the genetic makeup of the mother-infant dyad as well 

as the developmental state of the infant and environmental factors. While evidence to firmly 

support such hypothesis is currently lacking, what is clear is that we do not have a complete 

understanding of the consequences of increasing bOPN above the background level currently 

found in bovine milk-based IF. As such, evidence that rules out the potential adverse effects of 

exogenous and relatively high exposure to bOPN on maintenance or development of immune 

status in all infants does not currently exist. Because the assumption that bOPN are 

quantitatively bioequivalent to hOPN has not been proven, the appropriate levels of 

exogenously added bOPN which would not adversely alter the developmental trajectory of a 

given infant’s immune system have not been determined. Information provided in the notice 

and the amendment does not include study endpoints or other data and information that 

would be relevant to resolution of this potential issue. 

 

Specific questions related to the notifier’s GRAS conclusion 

Arla Foods provides the following overall rationale, in GRN 000716 as well as in amendments 

provided in response to our questions (see Appendix 1), for their conclusion that the intended 

use and use levels are generally recognized as safe: 

1) None of the toxicological studies, which included a rat acute toxicity study, rat 13-week 

repeat dose toxicity study, rat teratogenicity study, and genotoxicity/mutagenesis studies, 

nor other safety studies, which included three-month and six-month feeding studies in 

newborn rhesus monkeys and infants, respectively, showed adverse effects relevant to 

safety. Estimated dietary intake (EDI) from the intended use at the mean and 90th

percentile was calculated to be 24.8 and 39.5 mg/kg body weight (bw)/day, respectively. 

EDI was below the notifier’s deduced Acceptable Daily Intake (ADI) of 50 mg/kg bw/day, 

which was estimated based on No-Observed-Adverse-Effect-Level (NOAEL) of 2500 

mg/kg bw/day from a published rat teratogenicity study and the use of 50X safety factor. 

2) hOPN and bOPN are substantially similar. 

3) Human OPN is present in breastmilk. 

4) bOPN, present in cow’s milk, has been safely consumed throughout history. 
Page 4 - Administrative File, GRN 000716 

FDA’s viewpoints regarding each of the above provided assertions 

3⁄4 1. Absence of adverse effects in toxicological and other safety studies: 

The notifier states (in response to FDA’s Question 2): 

“The evaluation by the Expert Panel was that, based on the lack of toxicologically 

relevant adverse events in any of the safety studies conducted on OPN-10, the level 

of estimated intake at 50 mg/kg bw/day was safe for the intended consumers (i.e., 

infants).” 

FDA notes: 

a. The available evidence indicates that OPN is a bioactive substance with 

immunomodulatory and potentially pro-inflammatory properties. 

b. In a recent study, IF supplemented with 130 mg (OPN-10)/L (same as the intended 

use) was shown to increase some T-cell subpopulations, although the study did not 

appear to utilize experimental protocols designed to specifically evaluate 

activities/proportions of Th1/Th2 subpopulations (West et al., 2017). These 

observations are consistent with the presumption that bOPN in IF has 

immunological effects in infants, and do not resolve the question of whether bOPN 

supplementation of IF could have persistent effects on immune function by 

influencing the developmental trajectory of the immune system. The consequences 

of such effects would not necessarily be benign [i.e. susceptibility to proinflammatory diseases (Dietert, 2014)]. 

c. We also note that hOPN is strongly associated with development of immunemediated and inflammatory diseases (Boggio et al., 2016; Clemente et al., 2016). 

This association, though not a clear demonstration of causality, is consistent with 

other data and information on OPN’s effects. Furthermore, although clinical 

significance of the findings is not known, OPN has previously been identified as an 

autoantigen (Fierabracci et al., 1999; Merl et al., 2013). 

d. Safety assessment relies on toxicological studies that examine appropriate endpoints 

for the substances tested. None of the published safety studies discussed in the 

notice extensively evaluated its potential effect on immunotoxicity endpoints 

(DeWitt et al., 2012a, b). Thus a NOAEL from a rat teratogenicity study that was 

used to derive the notifier’s reported ADI may not accurately reflect OPN-10’s 

potential adverse effects on developmental immunotoxicity (DIT). Furthermore, as 

stated by the notifier, the use of a safety factor less than 100X can be justified based 

on appropriate scientific reasoning; however, without further context, it is not clear 

why a 50X safety factor is appropriate when the NOAEL is from a toxicological study 

that lacks relevant endpoints. Given the lack of these relevant endpoints in the 

studies cited, a safety factor of greater than 100X is not ruled out and may even be 

justified. 

e. For immunomodulatory substances, it is challenging to determine which study 

outcomes are pivotal and appropriate in the evaluation for the safety standard of 
Page 5 - Administrative File, GRN 000716 

“reasonable certainty of no harm.” Because of the nature of the immune network 

and its responses, some potential concerns cannot be addressed using standard 

toxicological endpoints within the currently accepted study designs. For example, a 

potential adverse effect of an altered immune response and pathology may only 

manifest itself later in life, upon which its causal link to bOPN exposure early in life 

would be difficult to prove. 

FDA’s evaluation of the relevance of the absence of adverse effects in 

toxicological and other safety studies: Given currently available data on 

bOPN and hOPN, endpoints from existing toxicology studies are not sufficient 

to resolve questions about potential adverse consequences of exogenous 

bOPN in IF on the developing immune system of infants. 

3⁄4 2. Similarities between human and bovine forms of OPN 

The notifier states (in response to FDA’s Question 2): 

“The OPN molecule contained in the Lacprodan OPN-10 product is substantially 

similar to the OPN molecule that is naturally found in human breast milk, and is 

being added to infant formula at a level not exceeding that found in breast milk.” 

FDA notes: 

a. This statement/conclusion infers that similarities at the amino acid sequence level 

between bOPN and hOPN indicate that bOPN is also “substantially” bioequivalent to 

hOPN and thus safe under the intended conditions of use. The existing literature is 

not consistent with the assertion that OPN from the two species are bioequivalent for 

the following reasons: 

x As stated by the notifier on pg. 25 of the notice, “[t]he diverse functions of OPN 

may be largely dependent on post-translational modification ...” According to 

Jiang & Lonnerdal, hOPN contains 34 phosphoserines, 2 phosphothreonines, and 

5 O-glycosylated threonine, whereas bOPN has 27 phosphoserines, 1 

phosphothreonines, and 3 O-glycosylated threonines. Furthermore, glycan 

structures between bOPN and hOPN are not the same (Christensen et al., 2012; 

Christensen and Sorensen, 2014). Therefore, it is not clear from the literature 

that either the specificity and/or the affinity of bOPN and hOPN to their 

interacting counterparts (i.e. receptors, such as integrins) are the same. 

x “Hydrolysis of OPN catalyzed by various proteases may have significant impact 

on functions of the peptides by changing binding affinities to distinct integrins” 

(Jiang and Lonnerdal, 2016). Accordingly, Christensen and Sorensen (2014) 

found several differences in cleavage patterns between human and bovine OPN. 

For example, whereas cathepsin D and plasmin can cleave hOPN at 7 different 

sites, only plasmin (without cathepsin D) cleavage was seen with bOPN. Whereas 

bOPN is cleaved mostly after Phe151, no preferential cleavage sites were identified 

for hOPN. Christensen and Sorensen conclude that: 

o “[t]hese differences can have significant effect on the ability of bOPN to 

interact with cells, as single amino acid differences next to the integrin-
Page 6 - Administrative File, GRN 000716 

binding motifs in the protein can have significant effects on its interaction 

with integrins ...” 

x As stated by the notifier, “intrinsically disordered proteins,” such as OPN, “exert 

biological functions by means of motifs and posttranslational modifications in the 

primary amino acid sequence ... and not dependent on any tertiary structure to be 

functional ...” (pg. 24). 

b. Rittling et al. (2014) showed that short peptides derived from digested bOPN (the 

test substance appears to be similar or perhaps identical to the subject of GRN 

000716) can enter systemic circulation resulting in unanticipated tumor suppressing 

activity likely due to regulation of blood vessel size. Furthermore, this activity can be 

pinpointed to short peptides derived from “SVAYGLK” sequence region 

(corresponding to “SVVYGLR” in hOPN) that interacts with several integrins. Given 

that there are two amino acid changes between bOPN and hOPN in this conserved 

orthologous sequence region, whether similar short peptides derived from hOPN will 

show a corresponding tumor suppressor/regulation of blood vessel size activity is 

not known. However, this study indicates the possibility that slight differences in 

primary sequence between bOPN and hOPN may result in systemic exposure of 

short peptides (produced upon digestion) that may have different and possibly 

unexpected or unintended bioactivities. This observation is of particular relevance 

for infant populations, given that infants have underdeveloped gastrointestinal 

anatomy and physiology compared to adults (Dallas et al., 2012; Lebenthal and Lee, 

1985), increasing the potential for systemic exposure to incompletely metabolized 

macromolecules via the oral route. 

c. Recently, Nielsen et al. (2017) reported that specific peptide fragments from hOPN 

were present in both foremilk and hindmilk samples from all four mothers tested, of 

which the majority appears to be derived from the C-terminal half of OPN. 

Importantly, many of these fragments from hOPN have significant amino acid 

differences compared to the corresponding orthologous bOPN fragments (see Fig. 2). 

As discussed above [2(a)], these subtle amino acid differences could result in 

differential bioactivities between peptide fragments from bOPN and hOPN. Given 

OPN-10, as stated by the notifier, consists of approximately 20% full-length and 80% 

N-terminal fragment (pg. 9 of the notice), it is not clear whether the proportion of 

full-length vs. N-terminal fragment of bOPN reflects similar proportion of specific 

OPN peptide fragments found in human milk. 

While it is likely that bOPN isolated from cow’s milk and hOPN in human milk share some 

common functionalities, there is substantial evidence in the literature that bOPN may have 

qualitatively and quantitatively different bioactivities compared to hOPN. In fact, 

Christensen and Sorensen (2014) have shown that “cleaved bOPN binds the important 

αVß3-integrin more competently than cleaved hOPN from human milk.” 

FDA’s evaluation of the relevance of the similarities between human and 

bovine forms of OPN: There is ample information in the published literature 

to indicate bOPN and hOPN undergo different post-translational and peptidic 

processing in vivo. There is further evidence that these differences in post-
Page 7 - Administrative File, GRN 000716 

translational processing of OPN likely influence its functionality. Thus, bOPN 

and hOPN cannot be assumed to be functionally bioequivalent. 

3⁄4 3. Presence of hOPN in human milk 

The notifier states that hOPN is present in human milk. However, the notifier also states in 

response to FDA’s Question 4: 

“Variation in the OPN content do[es] exist among mothers and among different 

geographical populations.” 

Furthermore, the notifier provides a statement from their expert (in response to FDA’s 

question 5) which states: 

“The degree of fragmentation is subject to large variation among individual 

mothers, which is most likely a reflection of the activities of proteases that cleave 

OPN in the most susceptible region around the thrombin/plasmin cleavage site.” 

FDA notes: 

a. Neither the published nor the unpublished4 studies discussed in the amendment 

have quantitatively distinguished the proportions of the various forms of hOPN in 

the human milk samples. Therefore, given the variability in expression as well as in 

proteolytic activity in human milk, it is not clear whether the proposed final 

concentration of 130 mg/L of OPN accurately reflects the “average” activity of hOPN 

in human milk. For example, high OPN levels detected via antibody-based assays 

(see below) in some human milk samples may in fact reflect much lower OPN 

biological activity due to extensive proteolysis in the sample. 

b. The published studies tested breastmilk OPN levels in limited samplings from only 

a handful of geographical locations. For example, estimation of the level of hOPN 

for the intended use was based on a single study (Schack et al., 2009) of 29 samples 

of breastmilk from Denmark, a country considered to have a relatively 

homogeneous population (Athanasiadis et al., 2016). As such, the notifier has not 

provided an adequate rationale to support the validity of the statistical assumptions 

needed to make inference from these studies to the general population outside of 

Denmark. Even if the new published studies with Asian mothers (Bruun et al., in 

press) are included in the future analyses, a rationale to justify assumptions for 

statistical inference would still be needed. 

c. Several studies report a wide variability of OPN expression (of which the proportion 

of fragmented and full-length forms are unknown) in human milk samples. Given 

the identification of several polymorphisms associated with increased OPN 

expression in humans (Chiocchetti et al., 2004), it may be argued that differences in 

OPN content in human milk samples simply reflect distinct 

 

4 Unpublished data mentioned in the amendment is now published online (Bruun et al., in press). 
Page 8 - Administrative File, GRN 000716 

polymorphisms/haplotypes of the mother without biological consequence. Higher 

levels of hOPN detected in human milk may even conceivably reflect the highest 

tolerable and/or safe level. However, published studies also strongly suggest that it 

is premature to conclude that high levels of hOPN are not associated with adverse 

health outcomes: 

x In humans, carriers of haplotypes B and C are associated with higher expression 

of OPN as well as higher risk for development of inflammatory immune disease 

(Clemente et al., 2016). 

x Expression levels of hOPN are correlated with obesity, diabetes, and 

inflammation (Kahles et al., 2014). 

x Leukocyte levels in breastmilk were found to depend on exposure of the mother 

and/or infant to pathogens, suggesting that the levels of pro-inflammatory 

cytokines such as OPN may reflect the health status of the mother-infant dyad 

(Hassiotou et al., 2013). 

d. The biological significance of different OPN levels in human milk is thus unknown. 

Furthermore, recent unpublished study (now published as Bruun et al., in press) 

showed that OPN levels decrease during the lactation period; as suggested by the 

authors, different levels of OPN may reflect the functional level appropriate for the 

developing infant. The above observation also support the hypothesis that the 

specific OPN levels may reflect the patho-physiological state of the mother and/or 

infant, or an adaptively appropriate level based on genetic, dietary, and 

environmental factors, and not merely due to inconsequential variation. Since it 

appears that neither the published nor the unpublished (now published as Bruun et 

al., in press) reports on OPN levels in breastmilk samples determined the potential 

relationship, if any, between the health-status of the mother/infant dyad and the 

levels of OPN, it is difficult to conclude that the appropriate levels of OPN for the 

intended use in the general population in the United States and elsewhere can be 

inferred from these studies. If the higher OPN levels were observed only in 

mothers/infants exposed to particular pathogens, which may only be prevalent in 

certain geographical locations, arithmetic mean from those studies may be skewed 

towards overestimation. Given that pathogen load/exposure and genetic 

polymorphisms are just some of potential confounders expected to influence the 

levels of hOPN in breastmilk, the appropriateness of use level based on arithmetic 

mean without context is questionable. 

e. There are several other sources of variability in hOPN levels. At any specific time 

point when the milk samples were collected, the measured level of endogenous 

hOPN may only reflect a level that was dictated by specific genetic, dietary, and 

environmental factors for an individual mother at that specific time point (Fields et 

al., 2016; Lee and Kelleher, 2016; Ruiz et al., 2017; Stam et al., 2013). Furthermore, 

how the temporal variation observed within individual mother-infant dyad 

accurately reflects temporal variations among populations of mother-infant dyads 

or vice versa cannot be inferred without further study. Finally, a biological response 

depends not only on the “dose” (i.e., concentration of OPN) but also on the affinity 

and the number of receptors/mediators expressed (i.e. integrins), which likely vary 

among individual infants. Thus, the aggregate mean concentration provides no 
Page 9 - Administrative File, GRN 000716 

information about the appropriate exposure in any particular case. Furthermore, if 

additional studies indicate that the level and form of hOPN secreted is intimately 

tied to the status of the individual mother-infant dyad, determining an appropriate 

level for the general population of bOPN for addition to IF becomes even more 

challenging. The comment from the notifier’s expert on the large variability of the 

degree of fragmentation observed among mothers suggests that there is a need to 

quantitatively evaluate the proportion of fragmented and full-length species in 

samples of human milk via methods other than ELISA assays, since ELISA assays 

(depending on the epitopes of the antibodies used) usually cannot distinguish 

between various proteolytic species of OPN. It is not clear from Schack et al. (2009) 

what the epitope(s) of the antibodies used was for their “in-house” developed ELISA 

assays; thus, whether their assays quantified all OPN species, including various 

proteolytic fragments, or only certain species is not clear. As stated above, because 

specific immunomodulatory bioactivity is influenced by proteolytic cleavage and 

post-translational modifications, it cannot be assumed that this variability is 

biologically neutral. There is currently insufficient generally available data to reach 

the conclusion that ~130 mg/L of OPN-10 (which consists of ~91 and ~39 mg/L of 

N-terminal fragment and full-length species, respectively) accurately reflects the 

“average” of levels and/or proportion of N-terminal vs. full-length species found in 

human milk samples. 

f. On Pg. 85 of the notice, the notifier states that“[b]ovine osteopontin is added to 

infant formulas in North and South East Asia and China at levels ranging from 21 

to 110 mg/L formula.” FDA’s view is that previous introduction of bOPN to IF in 

various parts of the world does not resolve our questions about the current 

consensus view among experts qualified by training and experience to assess the 

safety of this substance at the intended use level in IF given the existence of a large 

inter-individual variations in OPN concentration due to unknown factors (Bruun et 

al., in press; Nagatomo et al., 2004; Schack et al., 2009). 

FDA’s evaluation regarding the presence of hOPN in human milk: Although 

hOPN is present in human milk, there is considerable variation not only in 

the levels but also in specificity and proportions of various peptide fragments 

(i.e. full-length vs. various shorter peptides) among samples tested. Thus 

there is insufficient information in the published literature to conclude that 

the use of an arithmetic mean of 29 human milk samples from a single study 

(Schack et al., 2009) is an appropriate intended use level for all infants 

expected to consume IF. 

3⁄4 4. History of safe consumption of bOPN contained in cow’s milk 

The notifier states (in response to FDA’s Question 2): 

“... OPN from bovine sources has been consumed for centuries at low levels 

when contained in dairy products.” 

FDA notes: 
Page 10 - Administrative File, GRN 000716 

a) The relevance of the above statement to safety evaluation of exogenously added bOPN to 

IF at levels that far exceed those normally found in dairy products relies on the 

assumption that the intended subpopulation (i.e., infants) have “consumed for 

centuries” levels of bOPN that equal or exceed exposure from the notifier’s intended use 

of hOPN. According to Schack et al. (2009), the levels of bOPN in cow’s milk and cowderived IF are 18 μg/mL and 5.3-13.0 μg/mL, respectively. However, levels of bOPN 

from intended use is ~138 μg/mL. There is no evidence provided in the notice, or in the 

subsequent correspondence, that the intended subpopulation (i.e., infants) has been 

consistently exposed to bOPN at levels as high as ~138 μg/mL. Thus, occasional and/or 

lower exposure of bOPN from cow milk-derived foods and IF, as currently consumed 

during 0-6 months of infant growth, would not be expected to provide the same daily 

exposure that would result from the intended use (i.e., 39.5 mg/kg bw/day at the 90th

percentile as estimated by the notifier). Recognizing that hOPN exposures in human 

milk are currently poorly characterized but appear highly variable, and that intended 

bOPN use levels seek to replicate those exposures, significant increases in exposure raise 

the potential for new consequences not associated with historical exposure patterns. 

FDA’s evaluation of the history of safe consumption of bOPN contained in 

cow’s milk: Historical information in the published literature does not 

indicate that infants ages 0-6 months have consistently consumed bOPN at 

levels comparable to or above the intended use level. 

Additional data and observations considered by the FDA 

1) hOPN exists in a complex matrix within human milk (D'Alessandro et al., 2010; Munblit 

et al., 2017), and its overall activity may be modulated by other bioactive molecules, 

including immunoglobulins, maternal leukocytes and cytokines, present in human milk 

(Field, 2005). For example, OPN action may be counterbalanced by the modulation of 

OPN activity either directly (i.e., proteolytic and post-translational modifications) or 

indirectly (i.e. through other cytokines or other binding partners of OPN). It has been 

suggested that one of the important functions of human milk is to attenuate or 

counterbalance the pro-inflammatory response by newborns against pathogen exposure 

(Walker, 2010). Immune homeostasis is an important determinant in autoimmune and 

pro-inflammatory disorders (Chevalier et al., 2014; Humrich et al., 2010; Kollmann et 

al., 2017; Lenardo, 2003), and an adverse effect of an altered immune response and 

pathology may initially show only a subtle imbalance in immune cell subpopulations. 

Thus, hOPN in the context of human milk may have altered or attenuated biofunction 

when compared to bOPN exogenously added to IF for reasons other than species 

specificity. As Mundblit et al. (2017) states: 

“In view of the large number of potentially immune-active constituents in breast 

milk, investigation of only a limited range of constituents may well produce 

conflicting results. There is a lack of studies, attempting to assess [human milk] 

as a whole, rather than focusing on single components. In other words, the 

“soup” is likely more important than individual ingredients.” 
Page 11 - Administrative File, GRN 000716 

Thus, there is no a priori reason to assume that ~138 mg/L hOPN present in human 

milk has equal immunomodulatory or other bioactivity as ~138 mg/L of bOPN in IF 

from the intended use. 

2) It is well established that altering the levels of proteins that regulate biological response, 

such as hormones, cytokines and growth factors can cause major unintended biological 

effects. For example, inflammatory response to foreign pathogens must be kept in check 

in order to minimize tissue damage and/or sustained response (Murray and Smale, 

2012). One mechanism to achieve this is through a complex repression of genes 

involved in inflammation at the levels of signaling, transcription, post-transcription and 

translation, and processing. Given that OPN expression and activity is regulated at 

multiple levels (Christensen and Sorensen, 2014; Clemente et al., 2016; Lund et al., 

2009), exogenous exposure to bOPN could alter the appropriate balance and 

homeostasis of the immune components such as Th1/Th2/Th17 cells or 

myeloid/lymphoid cells (Kanayama et al., 2017). Relative to a default scenario of infant 

exposure to dynamically expressed hOPN in maternal milk, the risks and consequences 

of substantial (new intended use) versus minimal exposure to bOPN (i.e. exposure from 

current IF formulation) in the infant population are not necessarily equivalent. 

3) The notifier states (in response to FDA’s question 3): 

“It is also noted that Yamniuk et al. (2009) concluded that a complex between 

OPN and lactoferrin would likely be considered a benefit to the consumption of 

both OPN and lactoferrin, not a detriment; the authors suggested that ‘OPN may 

act as a carrier protein for LF [lactoferrin] in milk’.” 

However, as alluded to by Jiang and Lonnerdahl (2016), 

“Lactoferrin is a pleiotropic whey protein and is present at an even higher 

concentration than OPN in human milk, 1-10g/l ... [t]he possibility of a 

synergistic effect of lactoferrin and OPN has not yet been explored.” (emphasis 

added) 

Thus, if lactoferrin (LF) and OPN interactions involve “carrier-like” activities, then the 

actual concentrations of LF and OPN would be expected to influence the bioavailability 

of OPN. For example, the actual bioavailability of OPN at ~138 mg/L in IF may be 

different depending on the amount of LF that is present in IF. Furthermore, if such 

synergistic effects between LF and OPN do occur (there is no generally available data 

that suggests that they do not), then the ratio of [LF] to [OPN] in the formulation of IF 

may impact the immunomodulatory and other bioactive effects of OPN and LF. 

4) A recent publication on the safety assessment of food additives in IF proposed a 

“decision tree” to guide data requirements (Constable et al., 2017). According to this 

guide, standard toxicological datasets may not adequately address safety of OPN 

because of the high likelihood that immature immune system is a target organ for its 

actions. Regardless of the general acceptance of such decision tree for IF ingredient 

safety assessment, we continue to have questions about the likelihood of adverse 

unintended consequences resulting from the intended use of OPN which are not 

addressed by traditional toxicology tests or epidemiological and anthropometric-based 
Page 12 - Administrative File, GRN 000716 

clinical studies. Our concerns are based on the gaps in our understanding of (1) the 

functional role of OPN in breastmilk, (2) the significance of the inter-individual 

variability of the levels of OPN in breastmilk, (3) the uniqueness and developmentally 

dynamic nature of the infant immune system, (4) identifying safety endpoints relevant 

to assessing long-term effects of altering infants’ immune development, and (5) the 

relevance, if any, to safety assessment of a large body of scientific evidence that links 

OPN to pro-inflammatory diseases. As stated by Collinge et al. “[t]he difficulty in 

[endpoint] selection lies primarily in the gaps and limitations of the knowledge base and 

currently available methodologies for assessing [developmental immunotoxicity]” 

(Collinge et al., 2012); thus, FDA has not reached any conclusions on what potential 

toxicological and safety endpoints may be appropriate to conclude “reasonable certainty 

of no harm” for the intended use of bOPN at this time. However, FDA notes that the 

burden of proof for providing sufficient evidence that safety is generally recognized lies 

with the proponent. 

Overall conclusion by the FDA 

Our evaluation of the available data and information in the notice and in the scientific 

literature identified several questions about the intended use of bOPN described in GRN 716, 

given the properties of the substance and the target population. Our questions are based on: 

x The vulnerability of the specific population, including the potential to affect the rapidly 

maturing immune system and the potential for long-term unintended consequences 

x The lack of a comprehensive understanding of the modes of action of OPN and their 

consequences in the infant population 

x The absence of metrics or models for establishing the significance of existing bOPN and 

hOPN exposure to the intended bOPN exposure 

x The fundamental lack of clarity about whether or not absence of evidence of toxicity in 

standard toxicological tests is sufficient, or even relevant, to ensure reasonable certainty 

of no harm in this population given the known biological activities of bOPN; the notifier 

has not satisfactorily provided evidence that the “lack of evidence for adverse effects” 

sufficiently support “evidence of absence for adverse effects” 

x The apparent lack of consideration of these issues in the existing secondary literature 

x The absence of an evidentiary basis for the assumption that the presence of low levels of 

bOPN in current IF supports the safe use of bOPN at higher levels (138mg/L) 

The safety data discussed in the notice did not identify any adverse effects associated with the 

intended use of OPN-10 in nonexempt term IF at 138 mg/kg bw/day (final expected 

concentration of bOPN) at this time. However, given the available data and information about 

the properties of OPN and the infant immune system, we continue to have questions about 

whether the existing data is appropriate and sufficient to meet the reasonable certainty of no 

harm safety standard. These questions warrant further scientific investigation and robust 

public discussion (such as publication in peer-reviewed literature, scientific sessions at 
Page 13 - Administrative File, GRN 000716 

conferences, etc.) to determine whether scientists with specialized expertise in neonatal 

immunology would agree with other relevant food ingredient safety experts that the existing 

safety data presented by the notifier is adequate to establish reasonable certainty of no harm. 

GRN 000716 does not contain any evidence of the views of this expert community, either 

through inclusion of representative community members on the GRAS panel or via secondary 

literature.5 Until this evidence becomes available, FDA will continue to have questions about 

the safety of the intended use and the basis for general recognition of the safety data 

supporting the intended use of OPN-10 presented in GRN 000716. 

 

 

 _________________________ 

 Kotaro J. Kaneko 

 

 

5 For a discussion of the issues found in GRN 000716 from a general recognition perspective, as well as potential 

remedies, see GRN 000716 Policy Memorandum. 

Kotaro J. 

Kaneko -

S

Digitally signed by Kotaro J. 

Kaneko -S 

DN: c=US, o=U.S. Government, 

ou=HHS, ou=FDA, ou=People, 

0.9.2342.19200300.100.1.1=00

10342247, cn=Kotaro J. 

Kaneko -S 

Date: 2018.07.02 08:11:33 

-04'00'
Page 14 - Administrative File, GRN 000716 

Figure 1: “The role of osteopontin in immune cells focusing on the relationship between 

inflammation and apoptosis” [figure from (Iida et al., 2017)] 

 
Page 15 - Administrative File, GRN 000716 

Figure 2: Peptide fragments from hOPN identified in human milk samples and their 

corresponding locations within mammalian orthologous sequences 
Page 16 - Administrative File, GRN 000716 

References 

Adkins, B., Leclerc, C., and Marshall-Clarke, S. (2004). Neonatal adaptive immunity comes of 

age. Nat Rev Immunol 4, 553-564. 

Athanasiadis, G., Cheng, J.Y., Vilhjalmsson, B.J., Jorgensen, F.G., Als, T.D., Le Hellard, S., 

Espeseth, T., Sullivan, P.F., Hultman, C.M., Kjaergaard, P.C., et al. (2016). Nationwide 

Genomic Study in Denmark Reveals Remarkable Population Homogeneity. Genetics

204, 711-722. 

Basha, S., Surendran, N., and Pichichero, M. (2014). Immune responses in neonates. Expert 

Rev Clin Immunol 10, 1171-1184. 

Boggio, E., Dianzani, C., Gigliotti, C.L., Soluri, M.F., Clemente, N., Cappellano, G., Toth, E., 

Raineri, D., Ferrara, B., Comi, C., et al. (2016). Thrombin Cleavage of Osteopontin 

Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune 

Encephalomyelitis In Vivo. J Immunol Res 2016, 9345495. 

Bruun, S., Jacobsen, L.N., Ze, X., Husby, S., Ueno, H.M., Nojiri, K., Kobayashi, S., Kwon, J., 

Liu, X., Yan, S., et al. (in press). Osteopontin Levels in Human Milk vary Across 

Countries and within Lactation Period: Data from a Multicenter Study. Journal of 

Pediatric Gastroenterology and Nutrition Publish Ahead of Print. 

Chevalier, N., Thorburn, A.N., Macia, L., Tan, J., Juglair, L., Yagita, H., Yu, D., Hansbro, P.M., 

and Mackay, C.R. (2014). Inflammation and lymphopenia trigger autoimmunity by 

suppression of IL-2-controlled regulatory T cell and increase of IL-21-mediated effector 

T cell expansion. J Immunol 193, 4845-4858. 

Chiocchetti, A., Indelicato, M., Bensi, T., Mesturini, R., Giordano, M., Sametti, S., Castelli, L., 

Bottarel, F., Mazzarino, M.C., Garbarini, L., et al. (2004). High levels of osteopontin 

associated with polymorphisms in its gene are a risk factor for development of 

autoimmunity/lymphoproliferation. Blood 103, 1376-1382. 

Christensen, B., Klaning, E., Nielsen, M.S., Andersen, M.H., and Sorensen, E.S. (2012). Cterminal modification of osteopontin inhibits interaction with the alphaVbeta3-integrin. 

J Biol Chem 287, 3788-3797. 

Christensen, B., and Sorensen, E.S. (2014). Osteopontin is highly susceptible to cleavage in 

bovine milk and the proteolytic fragments bind the alphaVbeta(3)-integrin receptor. J 

Dairy Sci 97, 136-146. 

Clemente, N., Raineri, D., Cappellano, G., Boggio, E., Favero, F., Soluri, M.F., Dianzani, C., 

Comi, C., Dianzani, U., and Chiocchetti, A. (2016). Osteopontin Bridging Innate and 

Adaptive Immunity in Autoimmune Diseases. J Immunol Res 2016, 7675437. 

Collinge, M., Burns-Naas, L.A., Chellman, G.J., Kawabata, T.T., Komocsar, W.J., Piccotti, J.R., 

Shenton, J., and Wierda, D. (2012). Developmental immunotoxicity (DIT) testing of 

pharmaceuticals: current practices, state of the science, knowledge gaps, and 

recommendations. J Immunotoxicol 9, 210-230. 

Constable, A., Mahadevan, B., Pressman, P., Garthoff, J.A., Meunier, L., Schrenk, D., Speijers, 

G., O’Sullivan, A., and Hayes, A.W. (2017). An integrated approach to the safety 

assessment of food additives in early life. Toxicology Research and Application 1, 

2397847317707370. 

D'Alessandro, A., Scaloni, A., and Zolla, L. (2010). Human milk proteins: an interactomics and 

updated functional overview. J Proteome Res 9, 3339-3373. 

Dallas, D.C., Underwood, M.A., Zivkovic, A.M., and German, J.B. (2012). Digestion of Protein 

in Premature and Term Infants. J Nutr Disord Ther 2, 112. 
Page 17 - Administrative File, GRN 000716 

Denhardt, D.T., Giachelli, C.M., and Rittling, S.R. (2001). Role of osteopontin in cellular 

signaling and toxicant injury. Annu Rev Pharmacol Toxicol 41, 723-749. 

DeWitt, J.C., Peden-Adams, M.M., Keil, D.E., and Dietert, R.R. (2012a). Current status of 

developmental immunotoxicity: early-life patterns and testing. Toxicol Pathol 40, 230-

236. 

DeWitt, J.C., Peden-Adams, M.M., Keil, D.E., and Dietert, R.R. (2012b). Developmental 

immunotoxicity (DIT): assays for evaluating effects of exogenous agents on 

development of the immune system. Curr Protoc Toxicol Chapter 18, Unit 18 15. 

Dietert, R.R. (2014). Developmental Immunotoxicity, Perinatal Programming, and 

Noncommunicable Diseases: Focus on Human Studies. Adv Med 2014, 867805. 

Dowling, D.J., and Levy, O. (2014). Ontogeny of early life immunity. Trends Immunol 35, 299-

310. 

Field, C.J. (2005). The immunological components of human milk and their effect on immune 

development in infants. J Nutr 135, 1-4. 

Fields, D.A., Schneider, C.R., and Pavela, G. (2016). A narrative review of the associations 

between six bioactive components in breast milk and infant adiposity. Obesity (Silver 

Spring) 24, 1213-1221. 

Fierabracci, A., Biro, P.A., Yiangou, Y., Mennuni, C., Luzzago, A., Ludvigsson, J., Cortese, R., 

and Bottazzo, G.F. (1999). Osteopontin is an autoantigen of the somatostatin cells in 

human islets: identification by screening random peptide libraries with sera of patients 

with insulin-dependent diabetes mellitus. Vaccine 18, 342-354. 

Hassiotou, F., Hepworth, A.R., Metzger, P., Tat Lai, C., Trengove, N., Hartmann, P.E., and 

Filgueira, L. (2013). Maternal and infant infections stimulate a rapid leukocyte response 

in breastmilk. Clin Transl Immunology 2, e3. 

Humrich, J.Y., Morbach, H., Undeutsch, R., Enghard, P., Rosenberger, S., Weigert, O., Kloke, 

L., Heimann, J., Gaber, T., Brandenburg, S., et al. (2010). Homeostatic imbalance of 

regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus. Proc Natl 

Acad Sci U S A 107, 204-209. 

Hur, E.M., Youssef, S., Haws, M.E., Zhang, S.Y., Sobel, R.A., and Steinman, L. (2007). 

Osteopontin-induced relapse and progression of autoimmune brain disease through 

enhanced survival of activated T cells. Nat Immunol 8, 74-83. 

Iida, T., Wagatsuma, K., Hirayama, D., and Nakase, H. (2017). Is Osteopontin a Friend or Foe 

of Cell Apoptosis in Inflammatory Gastrointestinal and Liver Diseases? Int J Mol Sci 19. 

Jiang, R., and Lonnerdal, B. (2016). Biological roles of milk osteopontin. Curr Opin Clin Nutr 

Metab Care 19, 214-219. 

Kahles, F., Findeisen, H.M., and Bruemmer, D. (2014). Osteopontin: A novel regulator at the 

cross roads of inflammation, obesity and diabetes. Mol Metab 3, 384-393. 

Kanayama, M., Xu, S., Danzaki, K., Gibson, J.R., Inoue, M., Gregory, S.G., and Shinohara, M.L. 

(2017). Skewing of the population balance of lymphoid and myeloid cells by secreted 

and intracellular osteopontin. Nat Immunol 18, 973-984. 

Kollmann, T.R., Kampmann, B., Mazmanian, S.K., Marchant, A., and Levy, O. (2017). 

Protecting the Newborn and Young Infant from Infectious Diseases: Lessons from 

Immune Ontogeny. Immunity 46, 350-363. 

Lebenthal, E., and Lee, P.C. (1985). Gastrointestinal physiologic considerations in the feeding 

of the developing infant. Curr Concepts Nutr 14, 125-145. 

Lee, S., and Kelleher, S.L. (2016). Biological underpinnings of breastfeeding challenges: the 

role of genetics, diet, and environment on lactation physiology. Am J Physiol Endocrinol 

Metab 311, E405-422. 
Page 18 - Administrative File, GRN 000716 

Lenardo, M.J. (2003). Molecular regulation of T lymphocyte homeostasis in the healthy and 

diseased immune system. Immunol Res 27, 387-398. 

Lund, S.A., Giachelli, C.M., and Scatena, M. (2009). The role of osteopontin in inflammatory 

processes. J Cell Commun Signal 3, 311-322. 

Ma, N., He, Y., Xiao, H., Han, G., Chen, G., Wang, Y., Wang, K., Hou, C., Yang, X., Marrero, B., 

et al. (2014). BAFF maintains T-cell survival by inducing OPN expression in B cells. Mol 

Immunol 57, 129-137. 

Merl, J., Deeg, C.A., Swadzba, M.E., Ueffing, M., and Hauck, S.M. (2013). Identification of 

autoantigens in body fluids by combining pull-downs and organic precipitations of 

intact immune complexes with quantitative label-free mass spectrometry. J Proteome 

Res 12, 5656-5665. 

Munblit, D., Peroni, D.G., Boix-Amoros, A., Hsu, P.S., Van't Land, B., Gay, M.C.L., Kolotilina, 

A., Skevaki, C., Boyle, R.J., Collado, M.C., et al. (2017). Human Milk and Allergic 

Diseases: An Unsolved Puzzle. Nutrients 9. 

Murray, P.J., and Smale, S.T. (2012). Restraint of inflammatory signaling by interdependent 

strata of negative regulatory pathways. Nat Immunol 13, 916-924. 

Nagatomo, T., Ohga, S., Takada, H., Nomura, A., Hikino, S., Imura, M., Ohshima, K., and Hara, 

T. (2004). Microarray analysis of human milk cells: persistent high expression of 

osteopontin during the lactation period. Clin Exp Immunol 138, 47-53. 

Neal-Kluever, A., Aungst, J., Gu, Y., Hatwell, K., Muldoon-Jacobs, K., Liem, A., Ogungbesan, 

A., and Shackelford, M. (2014). Infant toxicology: state of the science and considerations 

in evaluation of safety. Food Chem Toxicol 70, 68-83. 

Renkl, A.C., Wussler, J., Ahrens, T., Thoma, K., Kon, S., Uede, T., Martin, S.F., Simon, J.C., and 

Weiss, J.M. (2005). Osteopontin functionally activates dendritic cells and induces their 

differentiation toward a Th1-polarizing phenotype. Blood 106, 946-955. 

Rittling, S.R., and Singh, R. (2015). Osteopontin in Immune-mediated Diseases. J Dent Res 94, 

1638-1645. 

Rittling, S.R., Wejse, P.L., Yagiz, K., Warot, G.A., and Hui, T. (2014). Suppression of tumour 

growth by orally administered osteopontin is accompanied by alterations in tumour 

blood vessels. Br J Cancer 110, 1269-1277. 

Ruiz, L., Espinosa-Martos, I., Garcia-Carral, C., Manzano, S., McGuire, M.K., Meehan, C.L., 

McGuire, M.A., Williams, J.E., Foster, J., Sellen, D.W., et al. (2017). What's Normal? 

Immune Profiling of Human Milk from Healthy Women Living in Different 

Geographical and Socioeconomic Settings. Front Immunol 8, 696. 

Schack, L., Lange, A., Kelsen, J., Agnholt, J., Christensen, B., Petersen, T.E., and Sorensen, E.S. 

(2009). Considerable variation in the concentration of osteopontin in human milk, 

bovine milk, and infant formulas. J Dairy Sci 92, 5378-5385. 

Shinohara, M.L., Jansson, M., Hwang, E.S., Werneck, M.B., Glimcher, L.H., and Cantor, H. 

(2005). T-bet-dependent expression of osteopontin contributes to T cell polarization. 

Proc Natl Acad Sci U S A 102, 17101-17106. 

Simon, A.K., Hollander, G.A., and McMichael, A. (2015). Evolution of the immune system in 

humans from infancy to old age. Proc Biol Sci 282, 20143085. 

Stam, J., Sauer, P.J., and Boehm, G. (2013). Can we define an infant's need from the 

composition of human milk? Am J Clin Nutr 98, 521S-528S. 

Walker, A. (2010). Breast milk as the gold standard for protective nutrients. J Pediatr 156, S3-

7. 

Wang, K.X., and Denhardt, D.T. (2008). Osteopontin: role in immune regulation and stress 

responses. Cytokine Growth Factor Rev 19, 333-345. 
Page 19 - Administrative File, GRN 000716 

West, C.E., Kvistgaard, A.S., Peerson, J.M., Donovan, S.M., Peng, Y.M., and Lonnerdal, B. 

(2017). Effects of osteopontin-enriched formula on lymphocyte subsets in the first 6 

months of life: a randomized controlled trial. Pediatr Res 82, 63-71. 

Zhang, X., Zhivaki, D., and Lo-Man, R. (2017). Unique aspects of the perinatal immune system. 

Nat Rev Immunol 17, 495-507. 

cc: GRN 000716

E-mail cc: HFS-255(SCarlson, RChanderbahn, MJDiNovi, RIMerker, SWestBarnette, 

PMGaynor, RShah, NBrewry, LShepherd) 

R/D:HFS-255: KJKaneko:12/15/17; 02/13/18; 02/14/18; 02/28/18; 03/05/18; 03/06/18; 

05/16/18; 06/20/18; 06/28/18 

Init:HFS-255: JFasano: 02/13/18; 03/05/18; 03/06/18; 05/16/18; 06/20/18; 06/28/18 

Init:HFS-255: SChoudhuri: 02/14/18; 03.05.18; 05/02/18; 06/20/18 

Init:HFS-255: RChanderbhan : 03/06/18 

Init:HFS-255: NBewry: 03/05/18 

Init:HFS-255: RShah: 03/05/18 

Edits/Init:HFS-255:SCarlson:4/26/2018; 06/19/2018 

F/T:HFS-255: KJKaneko: 06/29/18 
Page 20 - Administrative File, GRN 000716 

Appendix 1 

(FDA questions sent to the notifier) 
GRN000716 (Bovine whey-derived osteopontin)

 - FDA Questions and Comments -

Page 1 of 5

Date: October 3, 2017

Notifier: Arla Foods Ingredients Group PIS (AFI)

Chemistry 

1. The notifier describes a nitrogen quantification method to quantify the protein

content of their ingredient. However, the notifier also described other methods 

extensively within the notice, including the ELISA method. 

Please clarify what methods were used to quantify the protein content of their 

ingredient.

Toxicology 

2. On page 16, Table 4, the notifier states that the predicted bovine whey-derived 

osteopontin (OPN) exposure to infants <1 month of age from the intended use at the 

90th percentile is 39.5 mg/kg bw/day. On Pg. 86, the notifier states that the 

Acceptable Daily Intake (ADI) for bovine whey-derived OPN is 50 mg/kg bw/day 

based on No-Observed-Adverse-Effect-Level (NOAEL) of 2500 mg/kg bw/day from 

a published teratogenicity study in rats. However, traditionally, the safety factor for 

interspecies and intraspecies extrapolations using rodent studies is 100 (Benford, 

2000). Thus, the NOAEL of 2500 mg/kg bw/day would be extrapolated to ADI of 25 

mg/kg bw/day. 

Please provide a rationale for:

x Why the safety factor of 50, instead of 100, is appropriate.

x Why the estimated exposure of 39.5 mg/kg bw/day at the 90th percentile in a 

sensitive and vulnerable population is not a safety concern.

3. OPN is similar to lactoferrin in that they both possess immunomodulatory bioactive 

properties. It has been previously reported that lactoferrin binds OPN at 

approximately 3:1 ratio (Yamniuk et al., 2009). Lactoferrin is considered lower in 

non-supplemented infant formulas compared to breastmilk.

Given that many infant formulas do not supplement the formula with lactoferrin to 

levels normally observed in breastmilk, please provide a rationale as to why 

increasing the levels of OPN does not negatively impact the bioavailability of 

lactoferrin in bovine whey-derived OPN-supplemented infant formulas.

4. Estimation of the level of human OPN (hOPN) in breastmilk was based on a single 

study (Schack et al., 2009) of 29 samples from Denmark, a country considered to 

have relatively homogeneous population (Athanasiadis et al., 2016). As stated by the 
GRN000716 (Bovine whey-derived osteopontin)

 - FDA Questions and Comments -

Page 2 of 5

study authors as well as the notifier, there is also a considerable large variation in the 

level of OPN detected. 

Please address the following:

x Given the difference in demographics between nursing mothers in Denmark and 

the United States as well as the existence of large variations obtained from a 

small sample size, elaborate on why ~138 mg/L of OPN was chosen with respect 

to its level being generally recognized as safe. In your answer, elaborate on why 

the concentration of OPN (i.e. mg/L of breastmilk) was chosen rather than 

%OPN/total protein in breastmilk for the estimation of appropriate amount of 

OPN to be added to infant formula. 

x Given that one of the components in your safety narrative relies on the 

assumption that ~138 mg/L of OPN is the “normal” level of OPN found in all 

breastmilk across demographics and days post- parturition, it appears that the 

reliability of this information is vital to your assessment. If this is not the case, 

please elaborate.

5. Although ELISA quantitation described in Nagatomo et al. (2004) may be 

considered an overestimation, it appears that majority of hOPN in whey protein 

(presumably from crude preparations) in transitional and mature human milk is in 

the full-length form as assayed by Western blotting analysis using 10A16 monoclonal 

antibody (Fig. 2 of the manuscript). In fact, Bissonnette et al. (2012) confirmed the 

absence of cleaved hOPN form in breastmilk. However, the purified bovine wheyderived OPN in the notice consists mainly of cleaved peptides (80% C-terminal 

truncated vs. 20% full-length, pg. 9 of notice). Furthermore, as stated by 

Christensen and Sorensen (2014), “... the cleavage pattern observed for hOPN in 

milk is not necessarily identical to that for bOPN ... [k]knowledge of the exact 

cleavage sites is important, as small differences in the C-terminal of the 

fragments may have significant effects on the interaction between these and 

integrins. (emphasis added)” 

Please discuss why the potential differences in the proportion of full-length vs. 

cleaved peptide(s) between hOPN in human milk and bovine OPN (bOPN) in bovine 

milk are not a safety concern. 

6. On page 23, paragraph 4, and page 57, paragraph 3, there are blank parentheses 

after the citations. 

Please indicate whether this is a typo or missing references.

7. On page 79, in discussing findings of Lonnerdal et al. (2016), the notifier states:
GRN000716 (Bovine whey-derived osteopontin)

 - FDA Questions and Comments -

Page 3 of 5

“The decrease (P<0.05) plasma threonine concentration in the F130 group 

compared to the F65 group was not expected by the authors. The authors 

did not speculate on a reason for this slight, but significant change.”

Since the GRAS conclusion is made by the notifier, not the study authors, please 

clarify whether this “slight, but significant change” is a safety concern.

8. On page 22, in discussing the association of variant splice forms of OPN to cancer, 

the notifier states that the OPN-a form, a full-length native OPN present in human 

bovine milk, “has never been associated with such malignant properties.”

However, FDA’s literature search has identified two published reports (Blasberg et 

al., 2010; Hao et al., 2017) in which OPN-a form has been associated with non-small 

cell lung cancer:

Blasberg et al. concludes:

“OPNa overexpression was associated with increased bovine capillary 

endothelial tubule length and vascular endothelial growth factor secretion 

... These findings may lead to therapeutic strategies for selective isoform 

inhibition in non-small cell lung cancer.”

Hao et al. state:

“Collectively, our results have clearly demonstrated the clinical value of 

OPN-a in human non-small cell lung cancer as a potential target for 

therapy and a potential prognostic factor. The study has also revealed the 

importance of OPN-a in the aggressiveness of lung cancer cells with a 

particular relevance to bone metastasis related cell function of lung cancer 

cells.”

Please provide a brief explanation of why this information does not impact the 

notifier’s safety assessment.

References

Athanasiadis, G., Cheng, J.Y., Vilhjalmsson, B.J., Jorgensen, F.G., Als, T.D., Le 

Hellard, S., Espeseth, T., Sullivan, P.F., Hultman, C.M., Kjaergaard, P.C., et al.

(2016). Nationwide Genomic Study in Denmark Reveals Remarkable Population 

Homogeneity. Genetics 204, 711-722.
GRN000716 (Bovine whey-derived osteopontin)

 - FDA Questions and Comments -

Page 4 of 5

Benford, D. (2000). The Acceptable Daily Intake: A Tool for Ensuring Food 

Safety (ILSI Europe).

Blasberg, J.D., Goparaju, C.M., Pass, H.I., and Donington, J.S. (2010). Lung 

cancer osteopontin isoforms exhibit angiogenic functional heterogeneity. J 

Thorac Cardiovasc Surg 139, 1587-1593.

Christensen, B., and Sorensen, E.S. (2014). Osteopontin is highly susceptible to 

cleavage in bovine milk and the proteolytic fragments bind the alphaVbeta(3)-

integrin receptor. J Dairy Sci 97, 136-146.

Hao, C., Cui, Y., Hu, M.U., Zhi, X., Zhang, L., Li, W., Wu, W., Cheng, S., and 

Jiang, W.G. (2017). bovine whey-derived osteopontin-a Splicing Variant 

Expression in Non-small Cell Lung Cancer and its Effects on the Bone Metastatic 

Abilities of Lung Cancer Cells In Vitro. Anticancer Res 37, 2245-2254.

Nagatomo, T., Ohga, S., Takada, H., Nomura, A., Hikino, S., Imura, M., Ohshima, 

K., and Hara, T. (2004). Microarray analysis of human milk cells: persistent high 

expression of osteopontin during the lactation period. Clin Exp Immunol 138, 47-

53.

Schack, L., Lange, A., Kelsen, J., Agnholt, J., Christensen, B., Petersen, T.E., and 

Sorensen, E.S. (2009). Considerable variation in the concentration of osteopontin 

in human milk, bovine milk, and infant formulas. J Dairy Sci 92, 5378-5385.

Yamniuk, A.P., Burling, H., and Vogel, H.J. (2009). Thermodynamic 

characterization of the interactions between the immunoregulatory proteins 

osteopontin and lactoferrin. Mol Immunol 46, 2395-2402.

Additional Questions and Comments 

1. On page 18, the notifier states, “... would only be used in the wet blending-spray 

drying process of the production of infant formula, where ingredients are blended 

in water, homogenized, pumped to a heat exchanger for pasteurization, and then 

spray dried into a powdered product; for full- or near-full-term infants...”

Please clarify the meaning of “near-full term infants.” The notifier states that this 

ingredient is not intended for use in products that are preterm focused or exempt. 

2. On page 5 (A.2), the notifier states “OPN-10 contains at least 78% protein (N*6.38), 

greater than 95% of which is bovine whey-isolated OPN.” 

Please clarify what is the other 5% of protein. 
GRN000716 (Bovine whey-derived osteopontin)

 - FDA Questions and Comments -

Page 5 of 5

3. On page 5, the second paragraph, the notifier states that “...OPN is safe for human

consumption as a food ingredient in term nonexempt milk-based infant formula 

(which includes formula for infants 6-12 month of age)...”

Please clarify whether the ingredient will be added to non-exempt term infant 

formula for infants 0-12 months of age or only to non-exempt term formula for 

infants 6-12 months of age.

4. On page 45, first paragraph: Some of the cited references do not appear to support

the statements in this paragraph. The Greer reference only concerns premature 

infants; there is no information in this reference that addresses the amount of 

human milk that a term infant will consume daily. The information on the American 

Academy of Pediatrics (accessed September 1, 2015) website does not support the 

information provided in this paragraph. Additionally, we are unable to find the 

stated information in the US Environmental Protection Agency 2011 reference. The 

Butte 2005 reference appears valid. 

Please provide an accurate statement on the daily consumption of infant 

formula/human milk for term infants with appropriate references. 
August 3, 2017

Ray A. Matulka, Ph.D.

Director of Toxicology

Burdock Group

859 Outer Road

Orlando, FL 32814

Re: GRAS Notice No. GRN 000716

Dear Dr. Matulka:

The Food and Drug Administration (FDA, we) received Arla Foods Ingredients Group 

PIS (AFI)’s GRAS notice dated July 6, 2017. We received this notice on July 10, 2017, 

filed it as of the date of this letter, and designated it as GRN 000716.

The subject of the notice is bovine whey-derived osteopontin for use as a source of 

protein in milk-based term non-exempt infant formulas and in powdered beverages at 

levels up to 138 mg/L as consumed. The notice informs us of AFI’s view that this use of

bovine-whey-derived osteopontin is GRAS through scientific procedures. 

In accordance with 21 CFR 170.275(b)(1), the information in this notice described in 21 

CFR 170.225(c)(2) through (c)(5) will be accessible to the public at 

www.fda.gov/grasnoticeinventory. If AFI has any questions about the notice, contact me 

at 240-402-1007 or by electronic mail at Nadine.Bewry@fda.hhs.gov.

Sincerely,

Nadine Bewry, Ph.D., MPH

Division of Biotechnology

and GRAS Notice Review

Center for Food Safety 

and Applied Nutrition

Nadine N. 

Bewry -S

Digitally signed by Nadine N. Bewry -S 

DN: c=US, o=U.S. Government, ou=HHS, 

ou=FDA, ou=People, 

0.9.2342.19200300.100.1.1=0014360008, 

cn=Nadine N. Bewry -S 

Date: 2017.08.03 18:11:50 -04'00'
From: Cathryn Sacra <csacra@easconsultinggroup.com> 

Sent: Monday, November 05, 2018 10:39 AM

To: Morissette, Rachel <Rachel.Morissette@fda.hhs.gov>

Subject: RE: memo from pre-submission meeting on bovine lactoferrin in infant formula

Dear Rachel,

Our client has asked if it is possible to have a meeting to discuss the issues raised in the memo

before November 15th.

Best regards,

Cathryn

Cathryn W. Sacra

Director of Labeling and Cosmetic Services

EAS Consulting Group, LLC

1700 Diagonal Road

Suite 750

Alexandria VA, 22314

877-327-9808 (toll free) +1 571-447-5500 (main) +1 571-447-5505 (direct) 703-548-3270 (fax)

csacra@easconsultinggroup.com

www.easconsultinggroup.com

From: Morissette, Rachel <Rachel.Morissette@fda.hhs.gov> 

Sent: Tuesday, October 23, 2018 8:54 AM

To: Cathryn Sacra <csacra@easconsultinggroup.com>

Subject: memo from pre-submission meeting on bovine lactoferrin in infant formula

Dear Cathryn,

Please see attached our memo of meeting for the bovine lactoferrin pre-submission meeting held on

September 19, 2018. After you and your clients have had a chance to consider the points raised in the

meeting and clarified in this memo, we would strongly suggest scheduling a follow-up meeting with our

team, either in person or over the phone, to discuss the path forward. Please let me know if you have any

questions at this time.

Best regards,

Rachel

-------------------------------------------------------------

Rachel Morissette, Ph.D.

Consumer Safety Officer

 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety

U.S. Food and Drug Administration

rachel.morissette@fda.hhs.gov
MEMORANDUM OF MEETING (COR2018-6011)

Date: November 29, 2018

Time: 2:00 p.m. – 3:00 p.m. EST

Location: FDA, Center for Food Safety and Applied Nutrition, Office of Food Additive 

Safety, 5001 Campus Drive, College Park, MD 20740

Participants:

Visitors:

Cathryn Sacra EAS Consulting Group, LLC

Angela Walter Glanbia Nutritionals

Noreen Hobayan (by phone) Glanbia Nutritionals

Brent Peterson (by phone) Glanbia Nutritionals

Marian Kruzel, Ph.D. University of Texas Health Science Center

Robert Martin, Ph.D. Independent Advisor

Robin Guy Expert Consultant

CFSAN/OFAS/DBGNR:

Shayla West-Barnette, Ph.D. HFS-255

Jeremy Mihalov, M.S. HFS-255

Kotaro Kaneko, Ph.D. HFS-255

Jeremiah Fasano, Ph.D. HFS-255

Perry Wang, Ph.D. HFS-717

CFSAN/OFAS/DPR

Mical Honigfort, Ph.D. HFS-265

CFSAN/ONFL/IFMFS:

Andrea Lotze, M.D. HFS-850

Carrie Assar, Pharm.D. HFS-850

Suzanne Wolcoff, M.S., R.D. HFS-850

Subject: Pre-submission meeting for the intended use of bovine lactoferrin in infant and toddler 

formulas

In an electronic mail message dated November 5, 2018, Cathryn Sacra requested a meeting with 

OFAS/DBGNR to discuss issues that were raised during a meeting held on September 19, 2018. 

The subject of the previous meeting and the newly requested meeting was the intended use of 

bovine lactoferrin (bLf) in infant and toddler formulas, with a specific focus on the intended use 

in infant formulas.
The meeting started with OFAS/DBGNR noting that Ms. Sacra provided a memorandum dated 

October 10, 2018, summarizing the visitors’ perspective of the September 19, 2018 meeting.

OFAS/DBGNR noted that statements in that memorandum differed from those summarized in 

OFAS/DBGNR’s memorandum dated October 23, 2018. OFAS/DBGNR offered to clarify their 

perspective to assist the visitors in planning next steps.

The visitors summarized their conclusion that their intended use of bovine lactoferrin in infant 

formulas is GRAS. They discussed their expertise with respect to the safety of lactoferrin. They 

discussed publicly available literature they consider support the safety of bLF consumption by 

infants, including studies conducted using in vitro models and in infants. The visitors provided 

an overview of bLF’s iron-binding, antimicrobial, and immunoregulatory functions; the 

occurrence of lactoferrin in human milk, and the bioequivalence of bovine and human 

lactoferrins. The visitors discussed the bioavailability of bLF, stating that after consumption, bLF

does not cross the gut barrier or enter the bloodstream. 

In response to the visitors’ discussion of bLF’s antimicrobial properties, we stated that beneficial 

properties resulting from a substance’s consumption are not evaluated under the GRAS 

Notification Program. We stated that only data related to the safety of a substance’s consumption 

are evaluated under our program.

OFAS/DBGNR summarized our questions regarding limitations of the evidence of safety and the 

evidence of general recognition of safety supporting the visitors’ GRAS conclusion.

OFAS/DBGNR noted that previous GRAS conclusions for bLF in infant formula submitted to 

FDA through our GRAS Notification Program focused on relatively low use levels that were 

both broadly comparable to existing use levels in bovine milk-based infant formulas and 

significantly lower than levels of human lactoferrin (hLF) found in human milk. This fact pattern 

was a significant factor in our responses at the time. OFAS/DBGNR also noted that:

x lactoferrin exhibits complex functionality with respect to immune function,

x human lactoferrin and bLF are structurally related but not identical in properties or 

functionality,

x lactoferrin appears capable of exerting substantive effects via the oral route,

x the infant immune system is rapidly developing and sensitive to perturbation, and

x the studies presented in support of the notice do not incorporate endpoints relevant to the 

functionality of bLF.

OFAS/DBGNR stated that we acknowledge the absence of specific adverse effects (blood 

chemistry changes, body weight changes, tissue pathologies, etc.) following consumption of bLF

in the publicly available literature. However, we have questions about whether there continues to 

be consensus among qualified experts that the kind of studies and endpoints presented by 

Glanbia, in light of currently available information on the functionality of lactoferrin, are still 
accepted as appropriate and sufficient to establish the safety of the intended use level of bLF in 

infant formula. Specifically, our questions1 are:

x What is Glanbia’s basis for concluding that there is consensus, among scientists qualified 

by training and experience to assess the properties and activities of bLF in the context of 

the infant immune system, that no adverse effects will result from the use of bLF in the 

general infant population at the intended use level in infant formula?

x If the conclusion is based on the view that there are no relevant exposure-related 

qualitative or quantitative differences in bLF effects in infants between Glanbia’s current 

intended use level and use levels previously considered by FDA in GRAS notices, what 

is the basis for this view?

x If the conclusion is based on the view that none of the physiological effects generated by 

the properties and activities of bLF at the intended use level in this population are 

relevant factors in a safety assessment, what is the basis for this view?

x If the conclusion is based on the view that bLF and hLF are equivalent in their effects on 

infant physiology, what is the basis for that view?

OFAS/DBGNR provided the following recommendations2 for how the visitors can address our 

questions:

1) Determine and explicitly discuss Glanbia’s basis for concluding that currently available 

information about the properties and activities of bLF at the intended use level in this 

population is consistent with a conclusion of safety. This basis could involve one or more 

of the rationales identified above, supported by publicly available data and information.

2) Determine and explicitly discuss Glanbia’s basis for concluding that there is a consensus 

at this time among experts qualified to assess the safety of the intended use in infant 

formula that the data and information relied upon by Glanbia is appropriate and 

sufficient, Given the properties of bLF and the population, expertise in both immunology 

and pediatric development would be essential considerations. This basis could involve 

published secondary literature, statements from appropriate experts with views 

representative of the consensus in their fields, or the conclusions of a GRAS panel3 that 

incorporated the appropriate expertise. 

 

1 Subsequent to our meeting, we identified an additional issue. Given that: a) bLF differs with hLF in iron saturation; 

b) infants’ needs for exogenous iron differ developmentally, as well as individually; c) there appears to be debate

about iron homeostasis in infants younger than 9 months, what is the basis for concluding that bLF exposure 

resulting from the intended use would not be a safety concern?

2 We also recommend that Glanbia determine and explicitly discuss their basis for concluding that increased use 

levels of bLF will not adversely affect iron homeostasis in infants, given the currently available information in the 

literature.

3 Although not currently operative, FDA’s draft guidance on best practices for convening a GRAS panel is a useful 

illustration of the potential considerations involved in designing a GRAS panel capable of providing robust evidence 

of consensus.
OFAS/DBGNR clarified that we are not recommending that the visitors conduct or request new 

safety studies; we are recommending that the visitors develop a narrative that explicitly 

addresses our questions by placing the publicly available literature in the appropriate context.

OFAS/DBGNR concluded the meeting by agreeing to share this memorandum4 to clarify our 

questions.

__________________________________________

Shayla West-Barnette, Ph.D.

ATTACHMENTS:

1. List of attendees provided by Cathryn Sacra in an email dated November 26, 208.

2. Hard copy material provided by the visitors during the meeting on November 29, 2018.

3. Illustrative list of references 

 

4 In our view, recent literature suggests that discussion among the relevant scientific communities is beginning to 

shift from a focus on benefits alone to a broader consideration of the modes of action of LF and potential 

consequences for large-scale infant consumption of bLF at levels closer to those found in human milk. We have 

included an illustrative list of references that have informed our thinking on this topic. It is not intended to be 

comprehensive.

Shayla Westbarnette -S

Digitally signed by Shayla 

West-barnette -S 

Date: 2019.02.26 14:05:07 

-05'00'
R/D: HFS-255: SWestBarnette:11/30/2018

Edit/Comment: HFS-255:JFasano:12/20/2018

Edit/Comment: HFS-255:KKaneko:12/20/2018

Comment: HFS-255:SCarlson:12/20/2018

Edit: HFS-255:JMFasano:12/20/2018

F/T: HFS-255:SWestBarnette: 2/26/2019
From: Cathryn Sacra

To: West-Barnette, Shayla

Subject: RE: Pre-Submission Request for Bovine Lactoferrin

Date: Monday, November 26, 2018 3:58:45 PM

Attachments: image001.png

image013.png

Dear Shayla,

Below is a list of the attendees for the meeting on Thursday. All who are attending in person are US

citizens and will bring their driver licenses for identification. We will have several people who will

attend via conference call.

Glanbia Nutritionals

Angela Walter Senior Product Manager

Lactoferrin

Attending

Noreen Hobayan Director of Quality Assurance

Specialties

Conference Call/WebEx

Peter Budde Senior Director Product

Management Bioactive Dairy

Fractions

Conference Call/WebEx

Brent Peterson Senior Director

Ingredient/Bioactives R&D

Conference Call/WebEx

Ankur Jhanwar Senior Technical Services

Manager

Conference Call/WebEx

Marian Kruzel, PhD

University of Texas Health

Science Center at Houston,

Texas

Attending

EAS Consulting Group

Robert Martin, PhD Independent Advisor Attending

Robin Guy Expert Consultant Attending

Cathryn Sacra Director, Labeling and

Cosmetics

Attending

Cathryn W. Sacra

Director of Labeling and Cosmetic Services

EAS Consulting Group, LLC

1700 Diagonal Road

Suite 750

Alexandria VA, 22314

877-327-9808 (toll free) +1 571-447-5500 (main) +1 571-447-5505 (direct) 703-548-3270 (fax)







Illustrative References – Bovine Lactoferrin in Infant Formula 

Breakey, A.A., Hinde, K., Valeggia, C.R., Sinofsky, A., and Ellison, P.T. (2015). Illness in breastfeeding 

infants relates to concentration of lactoferrin and secretory Immunoglobulin A in mother's milk. Evol 

Med Public Health 2015, 21-31. 

Buccigrossi, V., de Marco, G., Bruzzese, E., Ombrato, L., Bracale, I., Polito, G., and Guarino, A. (2007). 

Lactoferrin induces concentration-dependent functional modulation of intestinal proliferation and 

differentiation. Pediatr Res 61, 410-414. 

Fernandez-Menendez, S., Fernandez-Sanchez, M.L., Gonzalez-Iglesias, H., Fernandez-Colomer, B., LopezSastre, J., and Sanz-Medel, A. (2017). Iron bioavailability from supplemented formula milk: effect of 

lactoferrin addition. Eur J Nutr 56, 2611-2620. 

Hare, D.J., Cardoso, B.R., Szymlek-Gay, E.A., and Biggs, B.A. (2018). Neurological effects of iron 

supplementation in infancy: finding the balance between health and harm in iron-replete infants. Lancet 

Child Adolesc Health 2, 144-156. 

Legrand, D. (2016). Overview of Lactoferrin as a Natural Immune Modulator. J Pediatr 173 Suppl, S10-15. 

Lonnerdal, B. (2010). Bioactive proteins in human milk: mechanisms of action. J Pediatr 156, S26-30. 

Lonnerdal, B. (2017). Development of iron homeostasis in infants and young children. Am J Clin Nutr

106, 1575S-1580S. 

Lonnerdal, B., Georgieff, M.K., and Hernell, O. (2015). Developmental Physiology of Iron Absorption, 

Homeostasis, and Metabolism in the Healthy Term Infant. J Pediatr 167, S8-14. 

Manzoni, P., Dall'Agnola, A., Tome, D., Kaufman, D.A., Tavella, E., Pieretto, M., Messina, A., De Luca, D., 

Bellaiche, M., Mosca, A., et al. (2018). Role of Lactoferrin in Neonates and Infants: An Update. Am J 

Perinatol 35, 561-565. 

Nguyen, D.N., Li, Y., Sangild, P.T., Bering, S.B., and Chatterton, D.E. (2014). Effects of bovine lactoferrin 

on the immature porcine intestine. Br J Nutr 111, 321-331. 

Villavicencio, A., Rueda, M.S., Turin, C.G., and Ochoa, T.J. (2017). Factors affecting lactoferrin 

concentration in human milk: how much do we know? Biochem Cell Biol 95, 12-21. 

Wessling-Resnick, M. (2017). Excess iron: considerations related to development and early growth. Am J 

Clin Nutr 106, 1600S-1605S. 
From: Cathryn Sacra

To: Morissette, Rachel

Subject: RE: pre-sub meeting memo on bovine lactoferrin

Date: Wednesday, October 10, 2018 5:13:26 PM

Attachments: image007.png

Glanbia - Memorandum of Meeting with CFSAN FINAL 10-10-2018.docx.pdf

Rachel,

Thank you for the update.  In the meantime, we have put together a Memorandum of Meeting with

our understanding of the issues that were raised in the meeting, which I have attached.  Please let

me know if you or the team have any comments or questions.

Best regards,

Cathryn

Cathryn W. Sacra

Director of Labeling and Cosmetic Services

EAS Consulting Group, LLC

1700 Diagonal Road

Suite 750

Alexandria VA, 22314

877-327-9808 (toll free) +1 571-447-5500 (main)  +1 571-447-5505 (direct) 703-548-3270 (fax)

csacra@easconsultinggroup.com

www.easconsultinggroup.com

From: Morissette, Rachel <Rachel.Morissette@fda.hhs.gov>

Sent: Thursday, October 04, 2018 9:15 AM

To: Cathryn Sacra <csacra@easconsultinggroup.com>

Subject: pre-sub meeting memo on bovine lactoferrin

Hi Cathryn,

I just wanted to let you know that I haven’t forgotten about sending you the meeting memo. We’re waiting

on a staff member to weigh in on the memo before I can finalize and send it to you. Hopefully within the

next week or so I should have it to you.

Best,

Rachel

-------------------------------------------------------------

Rachel Morissette, Ph.D.

Consumer Safety Officer

 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety

U.S. Food and Drug Administration

rachel.morissette@fda.hhs.gov

DBGNR Meeting Requests

Information for Logging into FARM/Appian

Requestor: 

Contact Information:

Date of Request:

Subject:

Date Request Received by DBGNR:

Date Prepared by DBGNR:

Prepared by:

If agent, name of company or individual this is on behalf of:

Cathryn Sacra, EAS Consulting Group csacra@easconsultinggroup.com 571-447-5505 August 13, 2018 (meeting to be held September 19, 2018) GRAS notice for bovine lactoferrin in infant formula August 13, 2018 August 20, 2018 Rachel Morissette