From: Morissette, Rachel

To: Cathryn Sacra

Subject: EAS/Glanbia meeting with FDA 11/29/18 - response

Date: Friday, May 10, 2019 11:47:00 AM

Attachments: image013.png

Dear Cathryn,

This is in response to your email of April 10, 2019. Shayla is currently on detail and asked me

to handle your request moving forward. We reviewed Glanbia’s response to the November 29,

2018 meeting memorandum that you sent and have the following comments below. In

addition, we strongly recommend that Glanbia FOIA the responsive records for GRN 000716

for the intended use of osteopontin in infant formula, another bioactive substance for which

FDA has similar questions regarding its safety. If you decide to submit a FOIA request, please

specifically request the amendments and correspondence for GRN 000716, including the

scientific memo, policy memo, and memorandum of meeting between CFSAN and the Center

for Biologics Evaluation and Research (CBER).

---------------

FDA notes that Glanbia has not satisfactorily explained why and how a GRAS conclusion can be

made for the use of high levels of bovine lactoferrin (bLF) based on a standard toxicological

testing paradigm. We note our concerns as follows:

Toxic chemicals exert their toxicity through interactions with their targets in the

cells/tissues causing cellular/tissue damage. Such tissue damage creates a pathological state

that can be detected through standard toxicological endpoint analysis. bLF is a bioactive

protein and not a toxic chemical. Safety issues related to immunomodulation do not create

a detectable pathological state and cannot be reliably addressed by simply investigating the

standard toxicological endpoints.

Furthermore, whereas homeostatic compensation may potentially buffer effects in adults

and reduce potential for long-term adverse consequences, this is less likely in the rapidly

developing and more sensitive infant population. This is true both for immunological effects

and for the additional issue [1] of iron storage and metabolism subsequently identified by

FDA.

Glanbia appears to misconstrue FDA’s questions about systemic effects, which do not relate

to systemic presence of bLF and associated food allergy concerns but rather systemic

immunomodulatory effects mediated either through local interactions with the gut or

through systemic entry via the relatively porous infant gut wall.

Glanbia presents anticipated benefits as evidence of safety, but beneficial effects cannot

compensate for potential risks in the same individual or in other individuals in the

population under FDA’s food ingredient safety assessment paradigm. Evidence of benefits
in a conventional food ingredient context can raise questions about concurrent risks arising

from the same modes of action, increasing the importance of clarifying all consequences

that may arise rather than simply arriving at a conclusion of net benefit.

Glanbia does not engage with the question of differential exposure in their reference to

previous GRAS notices. In arriving at a conclusion of no questions for these notices, FDA

relied in much greater part on widespread and longstanding exposure to roughly

comparable quantities of bLF in bovine milk-based infant formulas. However, as Glanbia

seeks to move up the dose-response curve for bLF and away from historical exposure

patterns to bLF in infant populations, FDA will have more questions about the mechanistic

and physiological basis for concluding that there is reasonable certainty that all effects will

be beneficial or neutral in all individuals. We are not aware of published studies that assess

dose-dependent effects of bLF on aspects of systemic immune function in infants. This

information by itself would not be sufficient to establish safety, but its absence significantly

hinders evaluation of the safety of this intended use.

Because the GRAS conclusion is Glanbia’s, it is up to them to identify or develop a safety

assessment strategy that incorporates the bioactivity and anticipated effects of bLF at this

exposure level in this population, using relevant biomarkers, analysis of modes of action and

their consequences, well-defined comparators, and other tools. If accepted by experts in the

field, this strategy, potentially a new paradigm for safety assessment, could provide a basis for

GRAS status for the intended bLF use.

The safety data presented by Glanbia does not reflect the physiological properties of bLF, the

anticipated effects in this population, or the relationship between exposure and effects. Based

on the current review of the published literature, FDA would expect to respond with a “‘no

basis” letter to any GRAS notice whose intended use involves bLF levels higher than use levels

proposed in previous GRNs, given the questions FDA has identified and the information

Glanbia has provided in response.

[1] FDA had previously raised questions about iron homeostasis based on available scientific

literature. Excess iron can have a variety of negative effects on development. The possibility

that higher levels of bLF in infant formula may alter iron homeostasis and storage and may

require changes in iron content was not adequately addressed by Glanbia in its response.

------------------

Until new data and information becomes available to address our safety concerns, we will

continue to question the basis for a GRAS conclusion for bLf at these use levels and do not feel

that further meetings to discuss Glanbia’s current GRAS conclusion would be productive. If

new data and information becomes available, please feel free to contact us again to discuss.

Best regards,
Rachel Morissette, Ph.D.

Regulatory Review Scientist

 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety

U.S. Food and Drug Administration

rachel.morissette@fda.hhs.gov

[1] FDA had previously raised questions about iron homeostasis based on available scientific literature. Excess iron

can have a variety of negative effects on development. The possibility that higher levels of bLF in infant formula may

alter iron homeostasis and storage and may require changes in iron content was not adequately addressed by

Glanbia in its response.
From: Cathryn Sacra

To: Morissette, Rachel

Subject: RE: EAS/Glanbia meeting with FDA 11/29/18 - response

Date: Thursday, June 13, 2019 11:38:07 AM

Attachments: image001.png

image013.png

Thank you, Rachel.  We really appreciate it.

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, June 13, 2019 11:28 AM

To: Cathryn Sacra <csacra@easconsultinggroup.com>

Subject: RE: EAS/Glanbia meeting with FDA 11/29/18 - response

Hi Cathryn,

I apologize for the delay. The FOIA office has changed our procedures and that has led to an unexpected

delay in your request. However, we are going to prioritize your request today and see that you receive the

documents hopefully by COB Friday. Again, I’m sorry for the delay.

Best regards,

Rachel

-------------------------------------------------------------

Rachel Morissette, Ph.D.

Regulatory Review Scientist

 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety

U.S. Food and Drug Administration

rachel.morissette@fda.hhs.gov
From: Cathryn Sacra <csacra@easconsultinggroup.com>

Sent: Monday, June 10, 2019 3:02 PM

To: Morissette, Rachel <Rachel.Morissette@fda.hhs.gov>

Subject: RE: EAS/Glanbia meeting with FDA 11/29/18 - response

Dear Rachel,

I hope that you are well.  I wanted to follow up on the recommendation that we put in an FOI

request for all the amendments and correspondence for GRN 000716, including the scientific memo,

policy memo, and memorandum of meeting between CFSAN and the Center for Biologics Evaluation

and Research (CBER).  We submitted that request on May 10th, but have not received the

information to date.  I know that your office would do the redactions, if any, so wanted to see if

there were any updates.

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: Friday, May 10, 2019 11:48 AM

To: Cathryn Sacra <csacra@easconsultinggroup.com>

Subject: EAS/Glanbia meeting with FDA 11/29/18 - response

Dear Cathryn,

This is in response to your email of April 10, 2019. Shayla is currently on detail and asked me

to handle your request moving forward. We reviewed Glanbia’s response to the November 29,

2018 meeting memorandum that you sent and have the following comments below. In

addition, we strongly recommend that Glanbia FOIA the responsive records for GRN 000716

for the intended use of osteopontin in infant formula, another bioactive substance for which

FDA has similar questions regarding its safety. If you decide to submit a FOIA request, please

specifically request the amendments and correspondence for GRN 000716, including the

scientific memo, policy memo, and memorandum of meeting between CFSAN and the Center
for Biologics Evaluation and Research (CBER).

---------------

FDA notes that Glanbia has not satisfactorily explained why and how a GRAS conclusion can be

made for the use of high levels of bovine lactoferrin (bLF) based on a standard toxicological

testing paradigm. We note our concerns as follows:

Toxic chemicals exert their toxicity through interactions with their targets in the

cells/tissues causing cellular/tissue damage. Such tissue damage creates a pathological state

that can be detected through standard toxicological endpoint analysis. bLF is a bioactive

protein and not a toxic chemical. Safety issues related to immunomodulation do not create

a detectable pathological state and cannot be reliably addressed by simply investigating the

standard toxicological endpoints.

Furthermore, whereas homeostatic compensation may potentially buffer effects in adults

and reduce potential for long-term adverse consequences, this is less likely in the rapidly

developing and more sensitive infant population. This is true both for immunological effects

and for the additional issue [1] of iron storage and metabolism subsequently identified by

FDA.

Glanbia appears to misconstrue FDA’s questions about systemic effects, which do not relate

to systemic presence of bLF and associated food allergy concerns but rather systemic

immunomodulatory effects mediated either through local interactions with the gut or

through systemic entry via the relatively porous infant gut wall. 

Glanbia presents anticipated benefits as evidence of safety, but beneficial effects cannot

compensate for potential risks in the same individual or in other individuals in the

population under FDA’s food ingredient safety assessment paradigm. Evidence of benefits

in a conventional food ingredient context can raise questions about concurrent risks arising

from the same modes of action, increasing the importance of clarifying all consequences

that may arise rather than simply arriving at a conclusion of net benefit.

Glanbia does not engage with the question of differential exposure in their reference to

previous GRAS notices. In arriving at a conclusion of no questions for these notices, FDA

relied in much greater part on widespread and longstanding exposure to roughly

comparable quantities of bLF in bovine milk-based infant formulas. However, as Glanbia

seeks to move up the dose-response curve for bLF and away from historical exposure

patterns to bLF in infant populations, FDA will have more questions about the mechanistic

and physiological basis for concluding that there is reasonable certainty that all effects will

be beneficial or neutral in all individuals. We are not aware of published studies that assess

dose-dependent effects of bLF on aspects of systemic immune function in infants. This

information by itself would not be sufficient to establish safety, but its absence significantly

hinders evaluation of the safety of this intended use.
Because the GRAS conclusion is Glanbia’s, it is up to them to identify or develop a safety

assessment strategy that incorporates the bioactivity and anticipated effects of bLF at this

exposure level in this population, using relevant biomarkers, analysis of modes of action and

their consequences, well-defined comparators, and other tools. If accepted by experts in the

field, this strategy, potentially a new paradigm for safety assessment, could provide a basis for

GRAS status for the intended bLF use.

The safety data presented by Glanbia does not reflect the physiological properties of bLF, the

anticipated effects in this population, or the relationship between exposure and effects. Based

on the current review of the published literature, FDA would expect to respond with a “‘no

basis” letter to any GRAS notice whose intended use involves bLF levels higher than use levels

proposed in previous GRNs, given the questions FDA has identified and the information

Glanbia has provided in response.

[1] FDA had previously raised questions about iron homeostasis based on available scientific

literature. Excess iron can have a variety of negative effects on development. The possibility

that higher levels of bLF in infant formula may alter iron homeostasis and storage and may

require changes in iron content was not adequately addressed by Glanbia in its response.

------------------

Until new data and information becomes available to address our safety concerns, we will

continue to question the basis for a GRAS conclusion for bLf at these use levels and do not feel

that further meetings to discuss Glanbia’s current GRAS conclusion would be productive. If

new data and information becomes available, please feel free to contact us again to discuss.

Best regards,

Rachel Morissette, Ph.D.

Regulatory Review Scientist

 

Center for Food Safety and Applied Nutrition

Office of Food Additive Safety

U.S. Food and Drug Administration

rachel.morissette@fda.hhs.gov
[1] FDA had previously raised questions about iron homeostasis based on available scientific literature. Excess iron

can have a variety of negative effects on development. The possibility that higher levels of bLF in infant formula may

alter iron homeostasis and storage and may require changes in iron content was not adequately addressed by

Glanbia in its response.
5500 Nobel Drive,

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Fitchburg, WI 53711

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www.glanbianutritionals.com

RESPONSE MEMORANDUM OF MEETING (COR2018-6011) PAGE: 1

DATE: Wednesday, 10 April 2019

SUBJECT: RESPONSE MEMORANDUM OF MEETING (COR2018-6011)

Dear Dr. West-Barnette,

This is in response to your email message dated February 26, 2019 in which you provided a memorandum of

the meeting that was held at FDA involving FDA representatives, Glanbia and EAS representatives on 

November 29, 2018. You also provided an attachment entitled “Illustrative References” which listed twelve 

references that your toxicologist considered to be important in ascertaining the safety of bovine lactoferrin in 

general and is of particular concern in infants who consume infant formulas. We note that three of the 

references are authored/co-authored by Dr. Bo Lonnerdal, who is one of the expert panel members on 

Glanbia’s GRAS Notice. Dr. Lonnerdal was also an expert panel member of GRN 669 as well.

We have also added an additional individual to the Expert Panel, Dr. Marian Kruzel, an internationally 

known immunologist. You may recall that Dr. Kruzel attended and participated in our November 29, 2018 

meeting. At that meeting, Dr. Kruzel gave a brief synopsis of the state of knowledge regarding lactoferrin. 

In fact, all sections of his narrative are included in this letter.

Your discussion of these issues is of great concern to us as we believe that bovine lactoferrin (bLf) is safe for 

use in infant formula (IF). In fact, we note that FDA has already approved six GRAS Notices for bLf; two of 

these GRNs (GRNs 465 and 669) are specific for the use of bLf in IF. As Glanbia has established that its bLf 

is equivalent to the bLf in the other GRNs, it stands to reason that these concerns are also applicable to the 

other GRNs as well.

We have placed our responses to the questions/issues that you raised directly below each question. We also 

note that the twelve references you provided were not tied to a specific question/concern that you raised. 

Where possible, we have cited the reference(s) that you provided and we believe pertains to that 

question/issue.

Also, at our meeting, you invited Glanbia to provide a copy of our responses for review prior to incorporating 

them into the document. We thank you for your review of our responses that we hope will satisfy your 

concerns. 

Additionally, we thank you for the opportunity to address the Agency’s five questions/concerns regarding 

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. We appreciate FDA’s input and statement of questions to help support the GRAS status of bLf at 

levels up to 1000 mg/L
5500 Nobel Drive,

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RESPONSE MEMORANDUM OF MEETING (COR2018-6011) PAGE: 2

1) 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?

Response: 

With respect to the use of bLf in infant formula, we note that FDA has already received, reviewed, and issued 

“good day” letters for two GRAS Notices (GRNs 465 and 669) for the use of bLf in IF. The GRAS Notice 

that was prepared by Glanbia contains essentially the same information and data that was used to support 

these IF GRAS Notices. The Expert Panel members chosen by Glanbia to review the GRAS determination 

are well recognized as experts in their respective fields of science and in Glanbia’s opinion, are well qualified 

to offer an opinion on the GRAS use of bLf in IF. The Expert Panel consists of world renowned experts, and 

most have published extensively in the field of lactoferrin, including an immunologist, a pediatric 

gastroenterologist, a professor of nutrition and internal medicine, a Dean with expertise in food science and 

human nutrition, a food regulatory advisor, and a toxicologist. In fact, two of the members also were 

involved in the preparation and review of GRN 669. Additionally, several recognized international 

regulatory bodies have also reviewed the use of bLf in IF and have determined that its use is safe in IF. In 

fact, they have reviewed the bLf produced by the same firms as were/are reviewed by FDA using essentially 

the same database. In addition, the use level of >600 mg/L was discussed and documented in GRN 669 even 

though the Notifier elected to use a lower level in one of their IFs. International regulatory bodies recognize 

the maximum, use level of 1000 mg/L. We are aware of at least one infant formula (IF) on the US market at 

this time that contains 600 mg/L and is currently on the market.

The intended use of Glanbia’s bLf in this GRAS Notice is for term infants using infant formula. These 

infants are presumed to be healthy at birth and are typically under the routine care of health professionals. 

The level of bLf in the IF is chosen to approximate the level that is found in mother’s breast milk which is 

considered to be the gold standard.

There is a large volume of scientific literature on lactoferrin, with consistent confirmations, for its safe use in 

IF. In addition, a number of papers also support the safe use at levels up to 1000 mg/L. While we note that 

active research is ongoing with regard to the use of bLf in IF, the prevailing consensus is that, based on the 

totality of the evidence currently available, the use of bLf in IF is safe when used as intended. 

The reference papers that you have provided support this conclusion. For example, in the reference by 

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, the authors offered the following statement (page 414): 
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RESPONSE MEMORANDUM OF MEETING (COR2018-6011) PAGE: 3

“Finally, our data also have practical implications. They indicate that bovine LF exerts effects on human 

intestinal cells that are similar to those induced by the human isoform. The comparative experiments showed 

that bovine isoform is even more potent than human LF in inducing cell growth and lactase expression. LF 

has been proposed for a number of therapeutic purposes in human disorders, including intestinal

inflammation, cancer prevention, and rotavirus infection (39– 41). Our findings add to this concept and 

suggest that bovine LF could be used as a functional component of infant formula to promote intestinal 

epithelial growth and differentiation. This effect is highly desirable, particularly in premature newborn infants 

or in intestinal diseases associated with epithelial atrophy.” 

We concur with the assessment that bLf exerts effects on human intestinal cells that are similar to those 

induced by the human isoform, and also note that this paper brings up the need for more research. 

In the reference paper by Fernandez-Menendez, S., Fernandez-Sanchez, M.L., Gonzalez-Iglesias, 

H., Fernandez-Colomer, B., Lopez-Sastre, J., and Sanz-Medel, A. (2017). Iron bioavailability from 

supplemented formula milk: effect of lactoferrin addition. Eur J Nutr 56, 2611-2620, the authors 

state as follows:

“Results 

The observed results with supplemented rats were compared with those found in rats receiving maternal 

feeding. Interestingly, differences were found between groups in iron for transport and storage compartments, 

but not in the functional one, depending upon the dose of iron administered and the chemical species.

Conclusion 

Considering the results obtained, supplementation with iron salts in excess of LF appears to be the best way 

of iron supplementation of formula milk.” (Page 2611)

The results of this study are further confounded as the authors further conclude:

“Finally, it should be noted to conclude that feeding rats with non-supplemented iron milk formula caused 

“latent iron deficiency”, that is, iron body stores became mildly depleted while the serum iron level dropped 

(even if, as stressed before, no change in the iron RBCs concentration was detected).

It is important to remark that the weight distribution of the animals among groups is different, despite that 

they were distributed into groups randomly. Differences in weight/development of the pups could result in 

differences in the demands and/or bioavailability of iron (that is, values observed in all the tissues and fluids 

analysed could not represent an accurate figure of the real bioavailability of iron from the supplement). In 

order to perform a statistical analysis in detail, further studies with larger number of individuals would be 

needed.” (page 2619)
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RESPONSE MEMORANDUM OF MEETING (COR2018-6011) PAGE: 4

Based on the results in this study, we conclude that this issue is not settled and additional research is needed.

In the reference paper by 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, they state as follows:

“Lactoferrin is one of the most represented and important bioactive proteins in human and mammal milk. In 

humans, lactoferrin is responsible for several actions targeting anti-infective, immunological, and 

gastrointestinal domains in neonates, infants, and young children. Evidence-based data vouch for the ability 

of supplemented lactoferrin to prevent sepsis and necrotizing enterocolitis in preterm infants and to reduce 

the burden of morbidity related to gastrointestinal and respiratory pathogens in young children. However, 

several issues remain pending regarding answers and clarification related to quality control, correct intakes, 

optimal schedules and schemes of supplementations, interactions with probiotics, and different types of milk 

and formulas. This review summarizes the current evidence regarding lactoferrin and discusses the areas in 

need of further guidance prior to the adoption of strategies that include a routine use of lactoferrin in neonates 

and young children.” (Page 561)

The authors’ conclusion is stated below:

“Conclusion

LF is a defense protein with diverse physiological functions. It has potent antimicrobial action against 

bacteria, fungi, viruses, and even some antibiotic-resistant strains. It has bacteriostatic, bactericidal, and antiadhesion effects. LF is gaining evidence for its role in neonatal and infant medicine, although several pending 

issues remain regarding quality of the commercial products, safety, schedules, and safety, warranting further 

studies.” (Page 564)

This paper indicates that there are open questions regarding lactoferrin that should be answered. We 

concur. Nevertheless, the use of lactoferrin as currently used is accepted as being beneficial.
5500 Nobel Drive,

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RESPONSE MEMORANDUM OF MEETING (COR2018-6011) PAGE: 5

In the review reference paper by Legrand, D. (2016). Overview of Lactoferrin as a Natural Immune 

Modulator. J Pediatr 173 Suppl, S10-15, the author’s stated conclusion is below:

“Conclusions

Lactoferrin is a unique molecule within the molecular arsenal of immunity, acting on the immune system 

both as a weapon and as a moderator. The many molecular and cellular targets of lactoferrin, and the complex 

and multi-parametric networks governing immune responses relative to the threat, make understanding the 

mechanisms of action of lactoferrin a challenge. The observation that dietary lactoferrin may mimic the 

protective and immune-modulating properties of endogenous lactoferrin strongly supports the hypothesis that 

lactoferrin may directly influence immune cells in the gut, very likely through cell receptors, resulting in 

systemic responses. Elucidating the exact way lactoferrin controls the Gut-Associated Lymphoid Tissue, the 

major lymphoid tissue in vertebrates, will provide the key to uncovering most mechanisms by which 

endogenous lactoferrin controls immunity. Such knowledge will also bring invaluable information to the

fine-tuning of infant milk formula development.”

We concur with the author’s conclusions in that additional research regarding open questions about 

lactoferrin is needed.

To summarize, it is widely accepted that there are open questions regarding lactoferrin including 

mechanisms of action, production, utility, etc. There are some published reports that need to be 

replicated to confirm the results. Some of the open questions are primarily theoretical and need to be 

discussed and vetted more thoroughly in open forums. It is the totality of the existing evidence 

which should be the determining factor in assessing the safety of lactoferrin at present. 

With this in mind, we offer a brief summary on lactoferrin structure and function below. 

Lactoferrin

Lactoferrin (LF) exhibits complex functionality with respect to immune function. It is a multifunctional ironbinding glycoprotein found in mammalian milk, tears, saliva, sweat, cerebrospinal fluid and neutrophils. It is 

secreted to all mucosal sites of mammals and is part of the innate immune response. By virtue of chelating 

iron, LF not only inhibits microbial growth but also reduces oxidative stress. It has been demonstrated that LF 

is able to reduce allergic response, and also protect against insult-induced mitochondrial dysfunction. While 

suppressing microbial growth, LF also directly exerts its first-line defense activity with significant impact on 

the development of adaptive immune responses by promoting the maturation of T-cell precursors into 

competent helper cells and by the differentiation of immature B cells into efficient antigen presenting cells. In 

addition, LF augments the delayed type hypersensitivity (DTH) response to antigens, leading to a strong 

induction of cell-mediated immunity (CMI). This summarizes what we know about functionality of LF. 
5500 Nobel Drive,

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RESPONSE MEMORANDUM OF MEETING (COR2018-6011) PAGE: 6

The wealth of reliable literature regarding the immune regulatory functions of LF is mostly derived from in 

vivo and in vitro observations when species-specific endogenous LF is tested in various experimental 

protocols. Based on these observations the functionality of LF has been well established in various 

physiological conditions. Endogenous LF (neutrophil-derived) undeniably plays an important role in 

mediation of complex cellular responses during the development of inflammation in relation to microbial 

infection, trauma, or other environmental insult (see Fig. 1). 

Figure 1 LF mediates cellular responses to accommodate physiological homeostasis. According to 

Kruzel et al. (Kruzel ML, Zimecki M, Actor JK. Lactoferrin in a context of Inflammation-Induced Pathology. 

Front Immunol. 2017 Nov 6;8:1438)

Briefly, injury defined by infection, or trauma leads to activation of the NF-κB signal transduction pathway 

within monocyte/macrophages and/or dendritic cells. This in turn stimulates the production of inflammatory 

mediators, which subsequently stimulates the production of fresh neutrophils and monocytes from bone 

marrow and activates circulating neutrophils to release various secondary mediators, including LF. By 

interacting with specific receptors on monocytes/macrophages and other immune and non-immune cells, LF 

attenuates inflammation and contributes to tissue repair and limits spread of infectious agents (Fig. 1). 
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RESPONSE MEMORANDUM OF MEETING (COR2018-6011) PAGE: 7

However, biological effects induced by oral administration of LF are different from the action of endogenous 

LF. Primarily, LF given orally helps to establish and maintain homeostasis of gastro-intestinal (GI) 

microbiota via safe and effective delivery of iron. It acts locally on intestinal epithelial cells before it is 

digested to small peptides and single amino acids. Both human and bovine LFs are recognized in a similar 

way by the mucosal-associated lymphoid tissue (MALT) in human gastrointestinal tract. Within the MALT, 

unique populations of dendritic cells interact with dietary antigens, and determine the fate of the resulting 

adaptive response, i.e., immunity versus tolerance. However, a portion of protease-resistant LF persists 

throughout the gastrointestinal tract providing local biological effects before it is excreted with feces (Dallas, 

Underwood, Zivkovic, & German, 2012). Subsequent to activation of immune cells in GI, lactoferrin induces 

various immune functions that are transduced systemically. Welch et al (Welsh KJ, Hwang SA, Boyd S, 

Kruzel ML, Hunter RL, Actor JK. Influence of oral lactoferrin on Mycobacterium tuberculosis induced 

immunopathology. Tuberculosis. 2011;91 Suppl 1:S105-13) reported that oral lactoferrin (supplied in 

drinking water) significantly reduced lung inflammation in mice infected with Mycobacterium tuberculosis. 

Similarly, Mohamed, et al., 2019 (Mohamed, W.A.; Salama, R.M.; Schaalan, M.F. 2019. A pilot study on the 

effect of lactoferrin on Alzheimer's disease pathological sequelae: Impact of the p-Akt/PTEN pathway. 

Biomed Pharmacother. 111, 714-723), demonstrated that LF, given orally, decreased serum IL-6 and 

increased serum IL-10 in patients who suffered from Alzheimer Disease, which led to a reduction of 

cognitive decline in these patients. 

In general, however, systemic effects of oral LF are not due to LF crossing the gut wall, but are a result of 

receptor-induced signal transduction. Therefore, both human lactoferrin (hLf) and bLf when administered 

orally provide similar biological effects on GI microbiota and activation of the mucosal epithelial cells 

including cell proliferation, differentiation, and expression of various signaling molecules.

According to Lonnerdal et al. (Bo Lönnerdal; Rulan Jiang;Xiaou Du; 2011. Bovine Lactoferrin Can Be Taken 

Up by the Human Intestinal Lactoferrin Receptor and Exert Bioactivities. Journal of Pediatric 

Gastroenterology and Nutrition. 53(6):606–614), bLf administered orally is taken up by the human lactoferrin 

receptor and exerts similar bioactivities as human LF on human colon epithelial cells such as induction of 

proliferation, differentiation, and TGFβ expression.

Finally, human milk-derived LF shares similar protein sequence, structure, and bioactivity with its bovine 

counterpart. Both human and bovine LFs have the same globular structure with two iron-binding sites on 

each lobe and an active N-terminal. Both are cationic proteins with isoelectric point (pI) around 8.5 and 

almost identical molecular weight as total amino acid composition is different only by two (2) amino acids 

(691 - human versus 689 - bovine). As the structure determines the function both human and bovine LFs are 

very similar in their functionality and should be considered bioequivalent.
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2) 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?

Response: 

Glanbia’s basis for determining that bLf is safe at levels of up to 1000 mg//L is based on the existing 

scientific data currently available. As Glanbia will be marketing its bLf to different IF manufacturers, it is 

possible that the manufacturer will choose a level that suits their purpose. In fact, there are IFs currently on 

the market that disclose various levels of bLf.

The mean daily intake of human lactoferrin for breast fed infants is approximately 1100 mg/day. It is the 

desire of some IF manufacturers to add bLf at levels that closely parallel the level in human breast milk.

We note that there are reports in the literature which discuss qualitative and quantitative differences on the 

effects of bLf in IF. In fact, several of the references that you provided discuss this in detail including 

beneficial effects and putative negative effects. Among these references were;

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.

These references point to the fact that there are confounding issues related to feeding IF, and for that matter, 

human breast milk. 

With regard to:

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;
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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. 

These reports all point to the fact that more research is needed to determine the effects of all bio-active 

compounds in milk to determine how they interact with other bio-active molecules, etc. At present, the 

determination as to how much of any individual compound may be necessary for a healthy infant is based on 

a case-by-case basis. 

There is a little unequivocal evidence that dietary LF can cross the gut wall intact and enter the hepatic portal 

system in physiologically relevant concentrations. In contrary, it was demonstrated that LF is acting directly 

on the GI mucosa by inducing epithelial cell proliferation, differentiation and expression of various signaling 

molecules. In this milieu, both human and bovine LFs are bioequivalent in a way that both are acting locally 

on brush border cells without crossing the gut wall barrier.

Biological effects of dietary compounds are relevant to the function of the GI tract as well as the total 

composition of the diet. It is worth mentioning that the diet is quickly changing over the first year of infant 

growth, including the introduction of solid foods, and it would be difficult to select the endpoints specific to 

LF but no other compounds of diet. The Johnston et al. (2015) and the Vaarala, et al. (2015) studies (see 

below) do discuss some specific endpoints that are discussed below.

The most relevant study for looking at the higher dose, is a double-blind, parallel-designed, gender-stratified 

prospective study (William H. Johnston, Claude Ashley, Michael Yeiser, Cheryl L. Harri, Suzanne I. Stolz, 

Jennifer L. Wampler, Anja Wittke and Timothy R. Cooper, 2015. Growth and tolerance of formula with 

lactoferrin in infants through one year of age: double-blind, randomized, controlled trial BMC Pediatrics 

15:173) 480 infants were randomized to receive a marketed routine cow’s milk-based infant formula 

(Control; n= 155) or one of two investigational formulas with bLf at 600 mg/L (n= 165) or 1000 mg/L (n= 

160) from 14–365 days of age. Investigational formulas also had a prebiotic blend of polydextrose (PDX) and 

galactooligosaccharides (GOS) and adjusted arachidonic acid (ARA). The primary outcome was weight 

growth rate from 14–120 days of age. Anthropometric measurements were taken at 14, 30, 60, 90, 120, 180, 

275, and 365 days of age. Parental recall of formula intake, tolerance, and stool characteristics was collected 

at each time point. Medically-confirmed adverse events were collected throughout the study period. The 

concentrations of bLf in the test formulas are within the range of LF concentration in human milk. 
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There were no group differences in growth rate (g/day) from 14–120 days of age; 353 infants completed the 

study through 365 days of age (Control: N =110; LF- at 600 mg/L N= 127; LF @ 1000 mg/L N= 116). Few 

differences in growth, formula intake, and infant fussiness or gassiness were observed through 365 day of 

age. Group discontinuation rates and the overall group incidence of medically-confirmed adverse events were 

not significantly different. From 30 through 180 days of age, group differences in stool consistency (P< 

0.005) were detected with softer stools for infants in the 600 mg/L and 1000 mg/L groups versus Control. 

Compared to the Control, infants who received investigational formulas with bLf and the prebiotic blend of 

PDX and GOS experienced a softer stooling pattern similar to that reported in breastfed infants. 

Vaarala et al. (Vaarala O, Saukkonen T, Savilahti E, Klemola T, Akerblom HK. 1995. Development of 

immune response to cow's milk proteins in infants receiving cow's milk or hydrolyzed formula. J Allergy 

Clin Immunol. Dec;96(6 Pt 1):917-23) showed that feeding infants with cow's milk-based formula induced 

systemic humoral and cellular responses to cow's milk proteins. However, T-cell response later declined, 

supporting the concept of oral tolerization to the T-cells.

The Johnston study demonstrated that routine infant formulas with the higher levels of bLf were safe, welltolerated, and associated with normal growth when fed to healthy term infants through 365 days of age. This 

combined with the Vaarala study that showed oral tolerization with regard to T-cells, in addition to a history 

of safe use, as described in the GRAS document, help support the safety of bLf at 1000 mg/L.

Indeed, a significant body of evidence from published intervention studies supports the safety of bLf for 

infants (GRN 669). In the 26 clinical trials identified in infants (from preterm and term at birth - 12 months) 

and in children (> 12 months) and involving approximately 4000 subjects, no adverse events related to the 

administration of bLf have been reported. The identified studies, completed in both healthy and vulnerable 

infants and young children, consistently report that bLf is well tolerated. As discussed above, the mean daily 

intake of human lactoferrin for breast fed infants is approximately 1100 mg/day. The level of bLf 

administered in these studies (up to 2,300 mg/day in term infants and up to 3,000 mg/day in children) 

adequately addresses the maximum predicted EDI's of bLf of this notification, and supports the safe use of 

bLf at 1000 mg/L for the intended uses. In addition, they show that both lower and higher concentrations of 

bLf are safe.
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The EFSA opinion (EFSA Panel on Dietetic Products Nutrition and Allergies (NDA). (2012b). Scientific 

opinion on bovine lactoferrin. EFSA Journal, 10 (5), 2701, [Available at: 

http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2012.2701/epdf)] includes the following:

“For infants with an age of 0 - 6 months, the applicant has estimated an intake of approximately 200 mg per 

kg bodyweight and 1.2 g bLF per day assuming that the mean intake is 1.2 liters of infant formula per day. 

The mean estimated intake of bLF by infants of 8 - 10 months of age would amount to 1.9 g per day. For 

adults, the applicant’s calculation estimates a mean and 95th percentile intake of 19 and 39 mg/kg 

bodyweight per day, respectively, and a mean and 95th percentile daily intake of about 1.4 g and 3.4 g, 

respectively.”

Therefore, based on the most recent evaluation of the publicly available data and observations, regarding 

bioequivalence of bovine milk derived LF and its human counterpart we believe that the total estimated 

intake up to 1000 mg/L of bLF is safe.

3) 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?

Response: 

We believe the references by Lonnerdal speaks to this issue.

Lonnerdal, B. (2010). Bioactive proteins in human milk: mechanisms of action. J Pediatr 156, S26-30.

“Although many factors are likely to be responsible for these differences, it is apparent that breast milk 

provides a multitude of bioactive proteins that are capable of physiological activities in the newborn infant 

and therefore can affect short- and long-term outcomes.” (page S26).

“...From this study, it cannot be ascertained that lactoferrin was responsible for the observed effect because 

there was no lactoferrin-only group. There is also a possible synergistic effect of lactoferrin and lysozyme as 

reported in an in vitro study by Ellison and Giehl.14 Lactoferrin has also been shown to have antiviral activity

against hepatitis C virus, cytomegalovirus, Herpes simplex virus, rotavirus, adenovirus, and human 

immunodeficiency virus.15

Three recent studies support that lactoferrin may prevent infections in children.” (page S26).
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In addition, we uncovered another article by Lonnerdal et al. and Bhatia which also addresses this issue.

Bovine Lactoferrin Can Be Taken Up by the Human Intestinal Lactoferrin Receptor and Exert 

Bioactivities, Lönnerdal, Bo; Jiang, Rulan; Du, Xiaou, Journal of Pediatric Gastroenterology and 

Nutrition: December 2011 - Volume 53 - Issue 6 - p 606–614 [Available at: 

https://journals.lww.com/jpgn/Fulltext/2011/12000/Bovine_Lactoferrin_Can_Be_Taken_Up_b

y_the_Human.8.aspx#pdf-link]. 

“Conclusions: CbLF is biologically active and is likely to exert several of the bioactivities of hLF if added to 

infant formula.” (page 606).

Bovine Lactoferrin, Human Lactoferrin, and Bioactivity, Bhatia, Jatinder, Journal of Pediatric 

Gastroenterology and Nutrition: December 2011 - Volume 53 - Issue 6 - p 589 [Available at: 

https://journals.lww.com/jpgn/FullText/2011/12000/Bovine_Lactoferrin,_Human_Lactoferrin,

_and.4.aspx#print-article-link]. 

“Bovine LF (bLF) inhibits the growth of a wide variety of bacteria, fungi, viruses, and parasites. Furthermore, 

a high homology between the human and bovine forms of LF suggests that supplementation of infant 

formulas with LF may provide similar protection against sepsis as observed with the use of human milk.”

(page 589).

Biological effects of dietary compounds are relevant to the function of the gastro-intestinal tract as well as 

total composition of the diet. 

A primary function of the GI tract is to digest dietary macromolecules and absorb the resultant nutrients into 

the hepatic portal system. Large proteins are first digested to peptides by gastric and pancreatic proteases, 

then are taken by peptidases present on the enterocytic brush border and broken down to smaller peptides and 

individual amino acids. Although the absorption of di-and tri-peptides is possible across the apical membrane 

in human GI, the mechanism of such absorption is still poorly documented. In general, there is little 

unequivocal evidence that dietary bioactive peptides can cross the gut wall intact in physiologically relevant 

concentrations (Miner-Williams WM, Stevens BR, Moughan PJ. 2014. Are intact peptides absorbed from the 

healthy gut in the adult human? Nutr Res Rev27:308–29). 
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However, in large studies on weaning piglets some of orally administered bLF was observed in the bile 

suggesting an active transport of LF by endocytosis via the epithelial cells into the bloodstream (Harada, E., 

Itoh, Y., Sitizyo, K., Takeuchi, T., Araki, Y., & Kitagawa, H.,1999). Characteristic transport of lactoferrin 

from the intestinal lumen into the bile via the blood in piglets. Comp Biochem Physiol A Mo/ Integr Physiol, 

I 24(3), 321-327). In general, however; the bioavailability of dietary peptides is very poor.

Again, human milk-derived LF shares similar protein sequence, structure, and bioactivity with its bovine 

counterpart. Both human and bovine LFs have the same globular structure with two iron-binding sites on 

each lobe and an active N-terminal. Both are cationic proteins with pI around 8.5 and almost identical 

molecular weight as total amino acid composition is different by two (2) amino acids (691 - human versus 

689 – bovine). As the structure determines the function both human and bovine LFs are similar in their 

functionality and should be considered bioequivalent.

Based on a long history of safe and beneficial utility of bovine milk in human nutrition it is conceivable that 

individual components of human and bovine milk are bioequivalent. Mostly, any effects are localized to the 

GI tract, with some being transduced systemically.

Although the primary function of the GI tract is to digest food to make the macromolecules easy to absorb, 

there is also an important immune aspect of GI function in humans. The GI tract, which is the largest 

immunologic organ in the body, is constantly exposed to an enormous array of exogenous antigens including 

commensal bacteria and ingested proteins. A single epithelial layer separates this antigenic load from the 

lymphocytes, antigen presenting cells (APC), stromal cells and other immune cells in the lamina propria that 

together comprise the mucosal-associated lymphoid tissue (MALT). Within the MALT, unique populations 

of dendritic cells (DCs) interact with dietary antigens, and determine the fate of the resulting adaptive 

response, i.e., immunity versus tolerance. According to Vaarala et al. (Vaarala O, Saukkonen T, Savilahti E, 

Klemola T, Akerblom HK. Development of immune response to cow's milk proteins in infants receiving 

cow's milk or hydrolyzed formula. J Allergy Clin Immunol. 1995 Dec;96(6 Pt 1):917-23), feeding infants 

with cow's milk-based formula induced systemic humoral and cellular responses to cow's milk proteins. Tcell response later declined, supporting the concept of oral tolerization. 

Human tolerance and safety of bLf has been established in a large number of intervention studies in infants 

(pre-term and VLBW, term) and young children (C. William H. Johnston, Claude Ashley, Michael Yeiser, 

Cheryl L. Harris, Suzanne I. Stolz, Jennifer LBMC Pediatr. 2015; 15: 173. Growth and tolerance of formula 

with lactoferrin in infants through one year of age: double-blind, randomized, controlled trial). The studies 

consistently report that the addition of bLf to formula or as a supplement was well tolerated, or that no 

adverse treatment-related effects were observed. Furthermore, the range of bLf safely consumed and tolerated 

in these studies is higher than the maximum predicted EDI’s of bLf in other reports (mean 1023 mg/day, or 

179 mg/kg BW/day, 90th percentile 1484 mg/day or 269 mg/kg/BW/day) in term infants aged 0 - 6 months) 

in term infants aged 0 - 6 months.
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It is important to emphasize that the mean daily intake of human lactoferrin for breast fed infants is 

approximately 1100 mg/day, which is higher than the bLf level proposed by Glanbia.

4) 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?

Response:

FDA has already approved six GRAS Notices for bLf; two of these GRNs (GRNs 465 and 669) are specific 

for the use of bLf in IF. As Glanbia has established that its bLf is equivalent to the bLf in the other GRNs, it 

stands to reason that these concerns are also applicable to the other GRNs as well. Nevertheless, we have 

included additional bioequivalent justification.

A primary function of the GI tract is to digest dietary macromolecules and absorb the resultant nutrients into 

the hepatic portal system. Large proteins are first digested to peptides by gastric and pancreatic proteases, 

then are taken by peptidases present on the enterocytic brush border and broken down to smaller peptides and 

individual amino acids. Human milk-derived LF shares similar protein sequence, structure, and bioactivity 

with its bovine counterpart. Both human and bovine LFs have the same globular structure with two ironbinding sites on each lobe and an active N-terminal. Both are cationic proteins with a similar isoelectric point 

and almost identical molecular weight as total amino acid composition is different only by two amino acids 

(691 - human versus 689 - bovine). As the structure determines the function, both human and bovine LFs are 

very similar in their functionality and should be considered bioequivalent, especially noting a long history of 

safe and beneficial utility of bovine milk in human nutrition.

As discussed earlier, both human and bovine LFs are bioequivalent in a way that both are acting locally on 

brush border cells without crossing the gut wall barrier.

This conclusion is also supported by the conclusion/statements made in the reports below:

Bovine Lactoferrin Can Be Taken Up by the Human Intestinal Lactoferrin Receptor and Exert 

Bioactivities, Lönnerdal, Bo; Jiang, Rulan; Du, Xiaou, Journal of Pediatric Gastroenterology and 

Nutrition: December 2011 - Volume 53 - Issue 6 - p 606–614 [Available at: 

https://journals.lww.com/jpgn/Fulltext/2011/12000/Bovine_Lactoferrin_Can_Be_Taken_Up_b

y_the_Human.8.aspx#pdf-link]. 

“Conclusions: CbLF is biologically active and is likely to exert several of the bioactivities of hLF if added to 

infant formula.” (page 606).
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Bovine Lactoferrin, Human Lactoferrin, and Bioactivity, Bhatia, Jatinder, Journal of Pediatric 

Gastroenterology and Nutrition: December 2011 - Volume 53 - Issue 6 - p 589 [Available at: 

https://journals.lww.com/jpgn/FullText/2011/12000/Bovine_Lactoferrin,_Human_Lactoferrin,

_and.4.aspx#print-article-link]. 

“Bovine LF (bLF) inhibits the growth of a wide variety of bacteria, fungi, viruses, and parasites. Furthermore, 

a high homology between the human and bovine forms of LF suggests that supplementation of infant 

formulas with LF may provide similar protection against sepsis as observed with the use of human milk.”

(page 1).

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.

“Finally, our data also have practical implications. They indicate that bovine LF exerts effects on human 

intestinal cells that are similar to those induced by the human isoform. The comparative experiments showed 

that bovine isoform is even more potent than human LF in inducing cell growth and lactase expression. LF 

has been proposed for a number of therapeutic purposes in human disorders, including intestinal

inflammation, cancer prevention, and rotavirus infection (39–41). Our findings add to this concept and 

suggest that bovine LF could be used as a functional component of infant formula to promote intestinal 

epithelial growth and differentiation. This effect is highly desirable, particularly in premature newborn infants 

or in intestinal diseases associated with epithelial atrophy.” (page 414).

Milk is a complex and complete source of bioactive molecules that help protect the newborn against 

infectious diseases and promote development while selectively enriching a protective and beneficial gut 

microbiota (Pacheco A.R., Barile D., Underwood M.A., Mills D.D. The impact of milk glycol biome on the 

neonate gut microbiota Annu Rev Anim Biosci. 2015 Feb 16; 3: 419–445). Epidemiological data suggest that 

human milk provides unique health benefits during early infancy that extend to long-lasting benefits.

Lactoferrin in particular is known to facilitate proper colonization of infant’s gut, and protect against diarrhea 

by preventing the attachment of enteropathogens in the gut (Ochoa Theresa, Cleary T.G. Effect of lactoferrin 

on enteric pathogens. Biochemie 2009, 91(1); 30-34). Although it may be difficult to assign specific 

functions for individual components of milk in development of infant’s immune system it is well established 

that LF is acting on the GI mucosa by inducing epithelial cell proliferation, differentiation and expression of 

various signaling molecules. In this context, both human and bovine LFs, as structurally similar proteins, 

exert their bioequivalent functions by acting locally on brush border cells without crossing the gut wall 

barrier. 
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Therefore, based on the most recent evaluation of the publicly available data and observations, regarding 

bioequivalence of bovine milk derived LF and its human counterpart we believed that the total estimated 

intake up to 1000 mg/L of bLf is safe.

5) 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? 

Response: 

The following five articles indicate that there are open questions regarding iron supplementation and status in 

infants as they mature to children and adults, and that more research is needed. We concur. Nevertheless, the 

use of naturally occurring higher levels of LF in nursing infants and exposures of lactoferrin in IF as currently 

used is accepted as being beneficial.

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.

In this study rats (N = 3/group) were randomly assigned to groups and it was determined that there was a 

difference in weight distribution in each dose group initially. Therefore, the study should be rerun with a 

larger N for statistical robustness.

The authors concluded in part, that the “Finally, it should be noted to conclude that feeding rats

with non-supplemented iron milk formula caused “latent iron deficiency”, that is, iron body stores became 

mildly depleted while the serum iron level dropped (even if, as stressed before, no change in the iron RBCs 

concentration was detected).”

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.

The authors state:

“What is clear from the evidence presented here is that little consensus exists on what the precise benefits 

and potential harms of iron supplementation are; numerous studies have identified a benefit in settings where 

iron deficiency anaemia is endemic, in terms of restoring haematological markers to those considered 

acceptable (panel 2), although the effects on neurodevelopment are not as obvious. Iron is crucial for 
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neurodevelopment, although direct intervention has become somewhat controversial, and will remain so until 

further large-scale longitudinal trials are able to categorically confirm or refute long-term benefits.” 

This reference points to the fact that there are confounding issues related to iron supplementation in IF. 

Lonnerdal, B. (2017). Development of iron homeostasis in infants and young children. Am J Clin Nutr 106, 

1575S-1580S. 

The authors concluded:

“Studies in human infants and experimental animals suggest that iron homeostasis is absent or limited in 

early infancy, which is largely due to a lack of regulation of the iron transporters

DMT1 and ferroportin. The high and unregulated absorption of iron in the newborn period may confer 

developmental benefits but raises the possibility of excessive iron accumulation.”

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.

“Iron is unique as there is no natural route for excreting excess iron. Thus, the possibility of 

overload certainly exists and is well known in adults. However, iron overload from orally provided 

iron as such has not been described in term human infants, and only implicated in premature infants 

with a known, or feared, consequence of increased iron-associated oxidative damage. Indications of 

excessive iron intakes by infants have been observed recently. As mentioned above, we noticed that 

supplementation of Swedish healthy, term breast-fed infants with iron drops caused decreased linear 

growth by 9 months of age (54). Since this adverse effect was not noted in Honduran infants, we 

hypothesized that the adverse effect was due to the iron-replete status of the Swedish infants. 

Indeed, when the Honduran cohort was divided into iron-replete and iron non-replete infants an 

adverse effect on growth was observed in the iron-replete group. A few other studies have also 

shown negative effects of iron supplements on growth (55, 56). However, in those studies the effect 

was noted for reduced weight gain rather than linear growth. It should be noted, though, that the 

nutritional status of the infants in those studies was compromised overall, which is known to 

decrease linear growth and cause stunting. Thus, when linear growth is compromised it is possible 

that the adverse effect of excess iron may be manifested differently and instead affects weight gain. 

However, a recent study on breastfed US infants given iron drops showed both a significant 

reduction in length gain and a trend towards reduced weight gain as compared to infants given ironfortified cereals (57). In the studies cited above, iron drops were given. 
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Since iron fortification is likely to result in less iron being absorbed, the potential risk of excess iron 

in this form may be lower. However, Lozoff et al (58) found that whereas infants with an initially 

low Hb (<106 g/L) benefitted from infant formula containing a higher level of iron (12.7 mg/L) and 

showed better developmental outcomes at 10 years of age than those given formula with less iron 

(2.3 mg/L) from 6 to 12 months of age, those with an initial Hb above 128 g/L showed worse scores 

when given formula with the higher level of iron. This suggests that an excess of fortification iron 

also may result in adverse effects.” 

Further studies are needed to explore the mechanism behind the adverse effect of excess iron.

Wessling-Resnick, M. (2017). Excess iron: considerations related to development and early growth. Am J 

Clin Nutr 106, 1600S-1605S.

The authors stated: “In conclusion, whether high iron affects erythropoiesis and, in particular, stress 

erythropoiesis is not clear. The influence of the iron sensor TfR2 on erythropoietin sensitivity may be 

relevant in iron-loading anemia. Ineffective erythropoiesis will evolve an anemic state that has been 

repeatedly shown to be detrimental to early development. Molecular studies to determine what steps in

erythropoiesis are sensitive to a high iron condition could provide insight into potential interventions. 

Similarly, despite our knowledge that several key micronutrients (e.g., vitamin A, copper, manganese, and 

zinc) support iron’s function in erythropoiesis, how these nutrients interact remains unknown to our

knowledge. It is necessary to consider many factors when formulating recommendations for iron 

supplementation because

these nutrient-nutrient interactions could possibly contribute to iron-induced toxicity. Research on mixtures 

of micronutrients can be carried out in both human and animal studies to establish a more comprehensive and 

holistic view of nutritional needs during pregnancy and early childhood."

Ochoa et al., 2012 (Ochoa, T. J., Pezo, A., Cruz, K., Chea-Woo, E., & Cleary, T. G., 2012. Clinical studies of 

lactoferrin in children. Biochem Cell Biol, 90(3), 457-467) showed no effect on the effects on iron status in 

infants fed bLf supplemented formula. 

One study by Chierici, et al. (Chierici, R., Sawatzki, G., Tamisari, L., Volpato, S., & Vigi, V. (1992). 

Supplementation of an adapted formula with bovine lactoferrin. 2. Effects on serum iron, ferritin and zinc 

levels. Acta Paediatr, 81(6-7), 475-479) showed that infants receiving the higher dose of bLf (100mg / 100 

ml) had significantly higher serum ferritin levels at days 90 and 150, while ferritin levels of breast-fed infants 

were significantly higher than in non-supplemented formula-fed infants at day 30 and day 90. 
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Davidsson, et al. (Davidsson, L., Kastenmayer, P., Yuen, M., Lönnerdal, B., & Hurrell, R. F. (1994). 

Influence of Lactoferrin on Iron Absorption from Human Milk in Infants. Pediatr Res, 35(1), 117-124) 

measured iron absorption in infants fed breast milk (with its native content of LF) and the same milk from 

which LF had been removed. Fractional iron absorption was significantly lower from breast milk than from 

LF-free breast milk. The authors concluded that the results do not support a direct role for LF in the 

enhancement of Fe absorption from human milk. 

Jenkins and Griffiths (Jenkins P and Griffiths J. 2014. Lactoferrin supplementation for very preterm infants. 

Infant 10 (5) 147-150. Accessed at: http://www.infantjournal.co.uk/pdf/inf_059_ent.pdf) of the ELFIN Trial 

Investigators Group stated because bovine lactoferrin does not bind strongly to the lactoferrin receptor in the 

human small intestine, it is not absorbed via the gastrointestinal tract and does not generate hypersensitivity 

or allergic immunological reactions.

More recently, Lonnerdal (Lönnerdal, B. (2016). Bioactive Proteins in Human Milk: Health, Nutrition, and 

Implications for Infant Formulas. J Pediatr, 173 Suppl, S4-9.) stated that the intestinal mucosa of breastfed 

infants is more developed than that of formula-fed infants. Increased mucosal development caused by 

lactoferrin may, therefore, increase the mucosal surface and not only enhance the uptake of iron but also of 

other nutrients.

The iron status of piglets fed control, antibiotic supplemented or bLf supplemented formula for 30 days was 

determined. Lactoferrin supplementation significantly increased serum iron values over controls by 22% and 

23%, on days 15 and 30, respectively, but did not affect serum total iron-binding capacity at either time point 

(Shan, T., Wang, Y., Wang, Y., Liu, J., & Xu, Z. (2007). Effect of dietary lactoferrin on the immune 

functions and serum iron level of weanling piglets. J Anim Sci, 85(9), 2140-2146).

There is a large volume of scientific literature on lactoferrin with reliable evidence for the safe use 

of bLf in IF at various levels. We are also aware that there are publications supporting the safe use 

of bLf at levels up to 1000 mg/L.

A significant body of evidence from published intervention studies supports the safety of bLf for infants 

(GRN 669). In the 26 clinical trials identified in infants (from preterm and term at birth - 12 months) and in 

children (> 12 months) and involving approximately 4000 subjects, no adverse events related to the 

administration of bLf have been reported. The identified studies, completed in both healthy and vulnerable 

infants and young children, consistently report that bLF is well tolerated. The mean daily intake of human 

lactoferrin for breast fed infants is approximately 1100 mg/day. The level of bLf administered in these studies 

(up to 2,300 mg/ day in term infants and up to 3,000 mg/day in children) adequately addresses the maximum 

predicted EDI's of bLf of this notification, and supports the safe use of bLf at 1000 mg/L for the intended 

uses.
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The EFSA opinion (EFSA Panel on Dietetic Products Nutrition and Allergies (NDA). (2012b). Scientific 

opinion on bovine lactoferrin. EFSA Journal, 10 (5), 2701. [Available at: 

http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2012.2701/epdf) includes the following, confirming in an 

exhaustive review that there were not any untoward effects of bLf at levels of 1000 mg/L:

“The toxicological information provided by the applicant included information from an in vitro genotoxicity 

study, a single dose study, and a four week and a thirteen-week oral repeated dose studies in rats. The Panel 

considers that bLF up to the highest dose (2,000 mg/kg bw per day) tested in this subchronic rat study did not 

show adverse effects which could be attributed to the test substance.

In an overall evaluation, the Panel considered that the novel food ingredient, bLF, is essentially a protein, a 

constituent of cow milk. According to the information provided by the applicant, bLF is present in the novel 

food ingredient (NFI) mostly as non-denatured lactoferrin. The Panel notes that lactoferrin is a normal 

constituent of human milk, and that the intended consumption of the bLF as specified in the application is 

within the levels of human lactoferrin consumed in breast milk by infants; human lactoferrin is also nondenatured.

The Panel notes that the mean estimated intake of bLF for infants up to the age of one year of approximately 

210 mg/kg bw per day would be around ten times lower than the highest dose (2,000 mg/kg bw per day) 

tested in a subchronic thirteen-week rat study, which did not show adverse effects related to bLF. For adults 

above 19 years of age the estimated intake is approximately 100 times lower. This maximum level of 

anticipated intake is considered a high intake scenario as opposed to a worst-case situation. The data provided 

suggest the absence of adverse effects of bLF at the proposed levels of consumption.”

Biological effects of dietary compounds are relevant to the function of gastro-intestinal tract as well as total 

composition of diet. It is worth mentioning that the diet is quickly changing over the first year of infant 

growth, and it may be difficult to select the endpoints specific to LF but no other compounds of diet. 

Therefore, in two studies relevant to LF functionality the endpoints are rather tox related than immune 

function. In addition, development of humoral and cellular immune responses to orally administered antigens 

in humans is poorly understood, although antigen administration has been suggested as a treatment for 

hypersensitivity disorders and autoimmune diseases. 

In a double-blind trial (Vaarala O, Saukkonen T, Savilahti E, Klemola T, Akerblom HK. 1995. Development 

of immune response to cow's milk proteins in infants receiving cow's milk or hydrolyzed formula. J Allergy 

Clin Immunol. Dec; 96 (6 Pt 1):917-23.), 10 infants received cow's milk-based formula, and 10 infants 

received a casein hydrolysate formula until the age of 9 months. Blood samples were taken at the ages of 6, 9, 

and 12 months. Cellular responses were assessed by proliferation assay of peripheral blood mononuclear cells 

to cow's milk proteins (beta-lactoglobulin, bovine serum albumin, and alpha-casein). Humoral responses to 

the same proteins were measured by ELISA for IgG antibodies.
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Feeding infants with cow's milk-based formula induced systemic humoral and cellular responses to cow's 

milk proteins. T-cell response later declined, supporting the concept of oral tolerization. Exposure to cow's 

milk proteins after the age of 9 months resulted in depressed cellular and humoral responsiveness to these 

proteins.

In other, double-blind, parallel-designed, gender-stratified prospective study (William H. Johnston, Claude 

Ashley, Michael Yeiser, Cheryl L. Harri, Suzanne I. Stolz, Jennifer L. Wampler, Anja Wittke and Timothy R. 

Cooper. Growth and tolerance of formula with lactoferrin in infants through one year of age: double-blind, 

randomized, controlled trial BMC Pediatrics (2015) 15:173) 480 infants were randomized to receive a 

marketed routine cow’s milk-based infant formula (Control; n= 155) or one of two investigational formulas 

with bLf at 0.6 g/L (LF-0.6;n= 165) or 1.0 g/L (LF-1.0;n= 160) from 14–365 days of age. Investigational 

formulas also had a prebiotic blend of polydextrose (PDX) and galactooligosaccharides (GOS) and adjusted 

arachidonic acid (ARA). The primary outcome was weight growth rate from 14–120 days of age. 

Anthropometric measurements were taken at 14, 30, 60, 90, 120, 180, 275, and 365 days of age. Parental 

recall of formula intake, tolerance, and stool characteristics was collected at each time point. Medicallyconfirmed adverse events were collected throughout the study period. The concentrations of bLf in the test 

formulas are within the range of LTF concentration in human milk. 

In conclusion, there were no group differences in growth rate (g/day) from 14–120 days of age; 353 infants 

completed the study through 365 days of age (Control: 110; LF-0.6: 127; LF-1.0: 116). Few differences in 

growth, formula intake, and infant fussiness or gassiness were observed through 365 day of age. Group 

discontinuation rates and the overall group incidence of medically-confirmed adverse events were not 

significantly different. From 30 through 180 days of age, group differences in stool consistency (P< 0.005) 

were detected with softer stools for infants in the LF-0.6 and LF-1.0 groups versus Control. Compared to the 

Control, infants who received investigational formulas with bLf and the prebiotic blend of PDX and GOS 

experienced a softer stooling pattern similar to that reported in breastfed infants. 

This study demonstrated that routine infant formulas with bLf were safe, well-tolerated, and associated with 

normal growth when fed to healthy term infants through 365 days of age.

Additional Supporting Publications

In addition to the above, we have undertaken to review other documents and reviews that speak to the use of 

bLf in IF. Below, we have inserted excerpts and conclusions from these papers to further support the fact that 

bLf at levels up to 1000 mg/L is safe and acceptable for use in IF. 
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In a review paper entitled “Structure and Functions of Lactoferrin as Ingredient in Infant Formulas”, Aly et 

al. [J Food Research, Vol 2, 25 – 36 (2013); Available at: 

https://www.researchgate.net/publication/263991247_Structure_and_Functions_of_Lactoferrin_as_Ingredien

t_in_Infant_formulas/download], reviewed the current status of lactoferrin in infant formulas. We have 

copied salient points from their discussion and conclusion below.

“In general, infant formulas have been designed to provide infants with all the required nutrients, being an 

adequate nutritional formula. For that purpose, infants with an age of 0-6 months, it has estimated to be safe 

an intake of approximately 1.2 g bovine lactoferrin per day from infant formula containing 200 mg bovine 

lactoferrin /100 g (European Food Safety Authority, EFSA, 2012). However, research advances are focused 

on those substances in human milk, which serve other than traditional nutritional roles. Attempts are in 

progress to supplement infant formulas with protective and trophic factors so far unique only to human milk. 

The final aim is not necessarily to mimic the composition of human milk in every respect, but to achieve 

physiological effects as in breast fed infants (Gallego, Pérez-Conesa, Bernal Cava, Periago-Castón, & Ros, 

2009). Since human milk contains a considerable amount of lactoferrin, special attention is paid to its 

functional role. Many of those functions are directly related to its ability to bind iron, that is, its effect on iron 

absorption and bacteriostatic and antioxidant activities. Based on this, the addition of lactoferrin to infant 

formulas seems to be reasonable; nevertheless, the supplementation of infant formulas should be discussed 

intensively because there has to be a scientifically proven advantage for the infant to get this protein by daily 

formula (Sawatzki, 1997). Recently, EFSA (2012) accepted and approved bovine lactoferrin as a new food 

ingredient. Nowadays, there are many infant formulas supplemented with lactoferrin available in the market 

(Mulder, Connellan, Oliver, Morris, & Stevenson, 2008). From results obtained by different authors, it can be 

concluded that the addition of lactoferrin, usually bovine, to infant formulas, does not affect iron absorption. 

However, given its ability to bind iron, its use in infant formulas could be useful for protecting the gut of 

infants against infections from microbial-requiring iron, its ability to reduce interelemental interactions and 

especially to protect infant formulas supplemented with iron and ascorbic acid against free radical formation.

In this context, Raiten, Talbot and Waters (1998) and Wakabayashi et al. (2006) reported that it is possible to 

enrich infant formulas with bovine or recombinant human lactoferrin, although the former does not seem to 

affect iron absorption, probably because of an incompatibility with the intestinal receptors, and in the latter, 

there is not enough available information to evaluate toxicity. In this regard, it must be taken into account that 

the enrichment of infant formulas with human lactoferrin would probably lead to an improvement in their 

amino acidic profile, making it more similar to that of human milk (Jovani, Barberá, & Farré, 2001). The 

EFSA (2012) considered that the bovine lactoferrin, is essentially protein constituent of cow milk and is

considered a novel food ingredient. Bovine lactoferrin is present in the novel food ingredient mostly as nondenatured lactoferrin. It must be noted that lactoferrin is a normal constituent of human milk, and that the 

intended consumption of the bovine lactoferrin is within the levels of human lactoferrin consumed in breast 

milk by infants; human lactoferrin is also non-denatured.”
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“Conclusion

The present review directs the attention towards some of the functional roles of lactoferrin and its roles in 

increasing the functional benefits of infant formulas. Lactoferrin is a new strategy for overcome some disease 

whether by orally administration or by food supplementation. Now is authorized and recommended using of 

lactoferrin as a new bioactive ingredient in the manufacturing of infant formulas to provide infants with 

nutritional and healthy effects especially for formula-fed infants and also after first 4-6 months. Many studies 

are required to study the effect of manufacturing and storage of infant formulas on lactoferrin. Also, it is 

possible using lactoferrin-derived functionally peptides for enrichment the infant formulas and this may be 

one of the growing and promising field of research.”

Even though the values sited in the paper are somewhat different than the EFSA doc, we concur with the 

authors’ assessment of the status of bLf.

We also call to your attention the research paper by Johnston et al. (Johnston, W.H., Ashley, C., Yeiser, M., 

Harris, C.L., Stolz, S.I., Wampler, J., Cooper, T.R. 2015. Growth and tolerance of formula with lactoferrin in 

infants through one year of age: Double-blind, randomized, controlled trial. BMC Pediatr 15(1):173. doi:10.l 

186/sl2887-015-0488-3) where they concluded as follows:

“Conclusions

Routine intact cow’s milk protein infant formulas with bLf at 0.6 and 1.0 g/L were associated with ageappropriate growth throughout the first year of life. This was the first large-scale pediatric nutrition trial in 

which formulas used concentrations of bLf that are within the range of Lf reported for mature human milk 

and included the prebiotic blend of PDX and GOS. Compared to infants who received the Control formula, 

infants who received investigational formulas with the prebiotic blend of PDX and GOS and bLf at 0.6 or 1.0 

g/L experienced a softer stooling pattern similar to that reported in breastfed infants. Consequently, this study 

demonstrated that routine infant formulas with bLf, a blend of PDX and GOS, and adjusted ARA were safe, 

well-tolerated, and associated with normal growth when fed to healthy term infants throughout the first year 

of life.”

We concur with the authors’ assessment.

In a publication entitled “Lactoferrin: A Critical Player in Neonatal Host Defense” (Telang S, 2018, 

Nutrients 10: 1228) reported on the role of lactoferrin infant nutrition. This paper discussed anti-microbial 

effects, immunomodulatory functions, efficacy, etc. of lactoferrin. The research is summarized as follows:
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“Abstract: Newborn infants are at a high risk for infection due to an under-developed immune system, and 

human milk has been shown to exhibit substantial anti-infective properties that serve to bolster neonatal 

defenses against multiple infections. Lactoferrin is the dominant whey protein in human milk and has been 

demonstrated to perform a wide array of antimicrobial and immunomodulatory functions and play a critical 

role in protecting the newborn infant from infection. This review summarizes data describing the structure 

and important functions performed by lactoferrin in protecting the neonate from infection and contributing to 

the maturation of the newborn innate and adaptive immune systems. We also briefly discuss clinical trials 

examining the utility of lactoferrin supplementation in the prevention of sepsis and necrotizing enterocolitis 

in newborn infants. The data reviewed provide rationale for the continuation of studies to examine the effects 

of lactoferrin administration on the prevention of sepsis in the neonate.”

The author’s conclusion supports the use of lactoferrin as stated below:

“Conclusions

Taken together, the experimental and pre-clinical studies examining the functions of Lf present

overwhelming evidence, supporting a pivotal role for this multifaceted glycoprotein in preventing infection, 

in immunomodulation, and bolstering host defense. Many questions remain to be answered regarding the 

function of this glycoprotein at the molecular level and the extent of direct and immune modulatory effects 

caused by supplementation of Lf in the diet. Several of these questions are best addressed by in vivo studies 

in patients. These are challenging studies, particularly as they are targeted towards the critical VLBW infant. 

However, the clinical data obtained thus far have been promising and certainly support the utility of 

continuation of studies to examine the effects of Lf supplementation on modulating the immune response and 

decreasing life-threatening infections in the highly vulnerable neonatal population. Several studies are 

currently underway, and their results will serve to clarify the benefits of Lf supplementation in the diet of the 

term and preterm infant, and potentially pave the way to using Lf in the clinical setting.”

We concur with the author’s conclusion.

In a publication entitled “Lactoferin in a Context of Inflammation-Induced Pathology”, Kruzel et al. (Kruzel 

ML, Zimecki M, Actor JK. 2017. Frontiers in Immunology (8): Article 1438; doi: 

10.3389/fimmu.2017.01438), the authors discussed the role of lactoferrin and concluded as follows:

“Conclusion

In conclusion, LTF plays a major functional role in physiologic homeostasis as related to development of 

disease and associated pathology. In many cases, LTF fulfills its anti-inflammatory roles via different cell 

receptors and activation of various cell signalling pathways, often through iron-dependent mechanisms. In 

fact, the ability of LTF to both sequester iron and to direct reactive oxygen intermediates is a major factor in 
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lessening damage due to excessive inflammatory responses. The immunomodulatory nature of this protein 

derives from its unique ability to sense the immune activation status of an organism and act accordingly.

The interaction of LTF with its receptors can trigger “redundant” protective effects as reflected by (1) 

regulation of enzyme activities and ROS production; (2) immune deviation and modulation; (3) change of 

cell phenotype and cytokine profile; (4) binding to LPS or competition with its receptors, and (5) prevention 

of cell apoptosis. Many additional immune pathways are also affected, which culminate in the consequence 

of attenuated pathological changes as tissue repair processes are initiated.”

We concur with the authors’ assessment of the current state of the art regarding lactoferrin.

There is a large volume of scientific literature on lactoferrin with reliable evidence for the safe use of bLf in 

IF . While we note that active research is ongoing in the area of bLf in IF, the prevailing consensus is that, 

based on the totality of the evidence currently available, the use of bLf in IF is safe when used as intended.

Therefore, there is consensus, among scientists qualified by training and experience, including those with 

years of experience investigating lactoferrin, 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. There are no robust studies that show that there are any effects on 

the immune system or iron status. Clinical studies repeatedly show that bLf has beneficial effects on infants 

throughout. Therefore, considering the totality of the existing evidence, we conclude that bLf is safe and 

GRAS for the uses intended by Glanbia. We also conclude that other scientists would reach the same 

conclusion.

We trust that our responses are satisfactory to your questions/concerns. Thank you for your cooperation and 

assistance.

Sincerely,

Angela Walter

Sr. Product Manager, Bioactive Dairy Fractions

Glanbia Nutritionals
From: West-Barnette, Shayla

To: Morissette, Rachel; Fasano, Jeremiah; Mihalov, Jeremy J.; Kaneko, Kotaro; Wang, Perry G

Cc: Carlson, Susan; Honigfort, Mical

Subject: FW: EAS/Glanbia meeting with FDA 11/29/18

Date: Wednesday, April 10, 2019 3:27:20 PM

Attachments: FINAL - GLANBIA RESPONSE TO FDA GRAS 9APR2019.pdf

Lactoferrin Pre-Submission Meeting 11-29-18 Final Transmittal Signed.pdf

Importance: High

Hello Everyone,

Glanbia provided responses to the questions we posed during our meeting with them last November

(please see attached). They request that we review their responses and then meet with them again.

Thank you,

Shayla

From: Cathryn Sacra <csacra@easconsultinggroup.com> 

Sent: Wednesday, April 10, 2019 1:45 PM

To: West-Barnette, Shayla <Shayla.WestBarnette@fda.hhs.gov>

Cc: Robert Martin <rmartin@easconsultinggroup.com>; Robin Guy

<RGuy@easconsultinggroup.com>; Walter, Angela <awalter@glanbia.com>

Subject: FW: EAS/Glanbia meeting with FDA 11/29/18

Importance: High

Dear Shayla,

I am attaching our response to the memorandum of meeting held on 11-29-18. During that

meeting, your team offered to meeting with us following our response to your questions. We are

available to meeting with FDA either by telephone or in person as soon as you have completed the

review of our response.

Best regards,

(b) (5)
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: West-Barnette, Shayla <Shayla.WestBarnette@fda.hhs.gov> 

Sent: Tuesday, February 26, 2019 1:29 PM

To: Cathryn Sacra <csacra@easconsultinggroup.com>

Subject: RE: EAS/Glanbia meeting with FDA 11/29/18

Hello Catherine,

By way of this message, I am sharing our memorandum from the meeting held on 11-29-18. I am

also sharing a document entitled, “Illustrative References”, which is an attachment to this

memorandum.

Thank you kindly for your patience with us.

Regards,

Shayla

From: Cathryn Sacra <csacra@easconsultinggroup.com> 

Sent: Monday, February 4, 2019 1:35 PM

To: West-Barnette, Shayla <Shayla.WestBarnette@fda.hhs.gov>

Subject: EAS/Glanbia meeting with FDA 11/29/18

Dear Shayla,

I hope you are well. I can’t imagine the state of your email inbox after 5 weeks, along with all the

projects that had to be put on hold and am sure that you are extremely busy. I did want to reach out

this week to see if there was any update to the memorandum of meeting from our meeting on

11/29/18. Our client, Glanbia, is very anxious to see the questions raised at the meeting so they can

be addressed and incorporated into the GRAS Notification on bovine lactoferrin.

Thanks so much,

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: West-Barnette, Shayla <Shayla.WestBarnette@fda.hhs.gov> 

Sent: Wednesday, December 12, 2018 11:53 AM

To: Cathryn Sacra <csacra@easconsultinggroup.com>

Cc: Walter, Angela <awalter@glanbia.com>

Subject: RE: Please Confirm Phone Participants from Yesterday's Meeting

Hello Cathryn,

We are writing our memorandum of the meeting and I plan to share this with you once it is

complete. The memorandum will include FDA’s questions for Glanbia.

Regards,

Shayla

From: Cathryn Sacra <csacra@easconsultinggroup.com> 

Sent: Wednesday, December 12, 2018 10:23 AM

To: West-Barnette, Shayla <Shayla.WestBarnette@fda.hhs.gov>

Cc: Walter, Angela <awalter@glanbia.com>

Subject: FW: Please Confirm Phone Participants from Yesterday's Meeting

Dear Shayla,

Thank you again for the meeting we had a couple of weeks ago. I wanted to follow up the questions

that FDA was putting together for the Glanbia team.

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: Cathryn Sacra <csacra@easconsultinggroup.com> 

Sent: Friday, November 30, 2018 2:51 PM

To: 'West-Barnette, Shayla' <Shayla.WestBarnette@fda.hhs.gov

Subject: RE: Please Confirm Phone Participants from Yesterday's Meeting

Dear Shayla,

The phone participants from Glanbia Nutritionals were:

Noreen Hobayan 

Brent Peterson 

Peter Budde

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: Cathryn Sacra 

Sent: Friday, November 30, 2018 2:18 PM

To: 'West-Barnette, Shayla' <Shayla.WestBarnette@fda.hhs.gov>

Subject: RE: Please Confirm Phone Participants from Yesterday's Meeting

Dear Shayla,

Thank you again for the meeting. I will verify who participated via conference call and get back to

you shortly.

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: West-Barnette, Shayla <Shayla.WestBarnette@fda.hhs.gov> 

Sent: Friday, November 30, 2018 12:18 PM

To: Cathryn Sacra <csacra@easconsultinggroup.com>

Subject: Please Confirm Phone Participants from Yesterday's Meeting

Hello Cathryn,

Thank you for coming to speak with us for yesterday’s meeting. I hope you found our

discussion to be helpful.

I am preparing the memorandum of meeting, and I want to be sure that I have the correct

attendee list. I have the following individuals listed as phone participants:

Noreen Hobayan 

Brent Peterson 

In the attendee list you sent to me by email before our meeting, you indicated that additional

participants would join by phone. It is possible that they joined, and that I did not see their

names on the WebEx list.

Can you confirm whether the names above were the only phone participants, or let me know

of any others who joined in by phone?

Thank you,

Shayla
Memorandum of Meeting

On Wednesday, September 19, 2018, representatives from Glanbia Nutritionals and EAS Consulting 

Group met with officials from CFSAN to discuss a draft GRAS notice for the use of bovine lactoferrin 

(bLf) in infant formula and toddler foods. The meeting took place at the CFSAN Office of Food Additive 

Safety, College Park, MD from 11:00 a.m. – 12 noon. The individuals attending the meeting are listed 

below.

Visitors

Glanbia Nutritionals 

Angela Walter, Senior Product Manager, Lactoferrin

Noreen Hobayan, Director of Quality Assurance, Specialties

Peter Budde, Senior Director Product Management, Lactoferrin

Brent Peterson, Senior Director, Ingredient/Bioactives R&D

Ankur Jhanwar, Senior Technical Services Manager

EAS Consulting Group

Cathryn Sacra, Director of Labeling and Cosmetic Services

Robin Guy, MS, DABT, RQAP-GLP

CFSAN Attendees

NOTE: A sign-in sheet was maintained. Regrettably, we did not get a copy of the sign-in sheet. Based 

on the introductions, we understood that the CFSAN representatives were from OFAS and the Infant 

Formula Group.

After introductions of the meeting attendees, Ms. Hobayan thanked the CFSAN representatives for 

arranging this meeting to allow Glanbia Nutritionals (Glanbia) and EAS to discuss their draft GRAS 

Notice for its use in infant formula and toddler foods and to seek advice and input from CFSAN as 

Glanbia planned to go forward on this project. This was followed by a brief introduction of Glanbia by 

Ms. Hobayan. Glanbia’s bLf (Bioferrin) was then discussed by Glanbia and EAS representatives to 

include: its formula, regulatory status, chemistry, exposure, and safety information. 

The meeting revolved around a PowerPoint presentation prepared by Glanbia and EAS that had been 

submitted to CFSAN in advance of the meeting. In advance of the meeting, Glanbia and EAS had 

indicated to CFSAN that there were two questions that they hoped to resolve and confirm at the meeting: 

1. Bioferrin is substantially equivalent to the bovine Lf’s that were previously reviewed by FDA for use 

in IF. Comparison of the specifications in table II-14 and infrared spectra in figures II-3a - II-3f listed on 

pages 34-38 of the draft GRN establish that Bioferrin is identical to the bLfs that have been approved by 

FDA.
2. That use levels up to 1000 mg/L is safe for use in infant formula as established by the opinions of 

other international regulatory bodies and FDA’s reviews of other bLf GRNs and is supported by the draft 

GRAS dossier.

There was good discussion related to the product. The CFSAN representatives essentially agreed that 

Bioferrin is equivalent to the bLfs that FDA had previously evaluated. Among the questions/issues raised 

by an OFAS toxicologist were:

• The FDA Toxicologist stated that the Agency has changed the way that they consider GRAS 

since GRN 669 for bioactive molecules. The Toxicologist stated that they want to make sure that 

there is a reasonable certainty of no harm, especially since this is for infants who inherently have 

an underdeveloped immune system. We note that Bioferrin in this GRN is intended for use in 

term infants who are presumed to be healthy at birth. Clarification is needed as to exactly what 

the toxicologist was referring to here.

• Most of the studies conducted have specific normal toxicological endpoints, especially with the 

studies conducted in adult rats. He implied that he didn’t know how these types of studies would 

be appropriate with regard to infant endpoints. He stated that he had not read the dossier; 

however, since the clinical trials were set up to address specific endpoints, we need to address 

how this relates to the infant, and how does this effect the preterm and VLBW infants. Again, as 

noted above, this is confusing as the GRN use is intended for term infants and not VLBW infants 

or premature infants, etc.

• He stated that even if EFSA found this use to be okay, we don’t know if they did the correct 

evaluations. 

• He wants the GRAS document to address:

The functionality of the protein

Intended population

Studies not looking at more subtle safety endpoints such as immunological effects.

As this was not clear to us, it was suggested that we contact the toxicologist and request any pertinent 

references that may have led to his concerns in this area.

The meeting ended at 12 noon. The visitors thanked the CFSAN representatives for their input.

Cathryn W. Sacra

Director, Labeling and Cosmetics

EAS Consulting Group
______________________________________________________________________________________________________________________________ 

Memorandum

Date March 21, 2019

From Jeremiah Fasano (HFS-255)

Through Shayla West-Barnette (HFS-255) _________________

Romina Shah (HFS-255) __________________

Kotaro Kaneko (HFS-255) __________________

Subject GRN 000716, Policy Memo

To Administrative File, GRN 000716

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 formulas for term infants is generally recognized as safe. On its face, this view seems 

plausible, in part due to the widespread consumption of both human osteopontin (hOPN1) in 

human milk and bOPN2 in bovine milk and milk-derived products. However, our evaluation

identified a number of questions involving potential adverse consequences of bOPN exposure 

1 hOPN is the product of the human SPP1 gene. 

2 bOPN is the product of the bovine SPP1 gene.

Kotaro J. 

Kaneko -S

Digitally signed by 

Kotaro J. Kaneko -S 

Date: 2019.03.25 

07:59:19 -04'00'

Romina 

Shah -S

Digitally signed by 

Romina Shah -S 

Date: 2019.03.25 

11:20:29 -04'00'

Shayla Westbarnette -S

Digitally signed by 

Shayla West-barnette -S 

Date: 2019.03.25 

12:42:13 -04'00'
Page 2 - Administrative File, GRN 000716

at the intended use level. These questions were prompted by what is currently known about the 

properties of hOPN, bOPN, and the infant immune system. In summary, the state of the 

science of the safety of bOPN as an ingredient is currently unsettled. We describe in detail 

these questions in the context of our interpretation of the current research literature in a 

science memo to the GRN 000716 file. The purpose of this policy memo is to detail our 

findings regarding the second element of GRAS, general recognition, as it pertains to the use of

OPN as an infant formula ingredient. 

The notifier’s basis for concluding that the intended use of bOPN is safe is described in the 

notice. None of the existing data and information contained in the notice, including traditional 

toxicology and safety studies ordinarily used to support food ingredient safety assessments, 

indicates toxicity. No adverse effects were reported in growth studies involving infants exposed 

to bOPN. Furthermore, given our understanding of protein-based toxicity, no such toxicity

would be predicted for bOPN. In addition, bOPN is a constituent of bovine milk, and is present 

in bovine milk-based infant formulas. Thus, there is an extensive history of exposure to this 

protein in this population, as well as adult exposure in a wide variety of milk-based products,

without any reported adverse effects in either population. Finally, human milk contains an 

orthologous hOPN with some structural and functional similarity to bOPN. Infants are exposed 

to this protein in the course of routine human milk consumption.

However, our review of the existing scientific literature generated a number of questions

(documented in the GRN 000716 science memo) about the intended use of bOPN that 

complicate the notifier’s narrative.3 First, we note that the intended use level of bOPN is 

roughly five to ten times higher than levels naturally present in bovine milk; and thus 

represents a significantly higher level of exposure than currently occurs in this population. We 

also note that the postnatal infant immune system is rapidly developing and that its typical 

developmental trajectory is susceptible to perturbation with unknown long-term 

consequences. OPNs have been reported to possess a variety of modes of action (MoAs), 

including immunomodulatory MoAs, with correlative and associative evidence indicating its 

potential roles in the etiology of certain adverse physiological conditions. Our review of the 

scientific literature indicates that while many studies have been conducted on bOPNs and 

hOPNs, these MoAs are still poorly and incompletely understood, especially in the context of 

exposure to infants. Furthermore, the functional differences and similarities between bOPN 

and hOPN, their significance for short-term immune status, as well as long-term health 

outcomes (if any) in this population are not well understood. Finally, the significance and 

consequences of observed interindividual variations in hOPN expression and exposure in the

context of genetic, dietary, and environmental impacts on maternal-infant dyad are not known. 

This is of particular importance because of the notifier’s reliance on the mean hOPN level from 

a single published study (Schenk et al., 2009) as the basis for a general use level in infant 

3 OFAS staff consulted colleagues elsewhere in CFSAN with relevant expertise, including a board-certified 

neonatologist, as well as colleagues at FDA’s Center for Biologics Evaluation and Research with expertise in 

assessment of biologically active proteins and immunomodulation, as described in Attachment 1 (“Intercenter 

Discussion on Osteopontin”). The focus of discussion was not on developing answers to the questions we had 

identified, but rather on whether such questions were reasonable when considering the basis for a conclusion of 

general recognition. The results of our discussion suggest that there may not be a robust consensus at this time 

about the appropriate and sufficient data needed to establish safety for ingredient uses of this kind.
Page 3 - Administrative File, GRN 000716

formula. We note that the number of subjects who donated milk in the study was not large and 

was seemingly from a relatively homogeneous population. At this juncture, it is not clear from 

the literature whether or not derivation of a target value for intended use can be justifiably 

inferred for the entire population by estimating a single arithmetic mean from one or several 

studies with potentially unrepresentative sampling, or indeed whether a single mean value is 

appropriate given the observed variation in expression and anticipated activity of OPN.

In our view, these questions are consistent with the recommendations of the Institute of 

Medicine4 who suggested that, in additional to traditional toxicological endpoints, the 

“appropriate level of [safety] assessment” for a new ingredient for infant formula use consider:

x The reversibility of potential harmful effects,

x The severity and consequences of adverse effects,

x The time of onset of manifestations of the adverse effects,

x The likelihood that a new ingredient could adversely affect a specific system, and

x Whether the effect would be common or rare.

We initially attempted to resolve these questions by the routine approach of asking clarifying 

questions of the notifier. However, our review of the responses to these questions convinced us 

that they could not be resolved by additional data in any reasonable time frame. Further 

consideration indicated a basic question underlying our earlier inquiries; namely, what is the 

significance of the questions we identified? Complete mechanistic characterization of a 

substance in the organism is rarely, if ever, necessary to reach a conclusion of safety. On the 

other hand, advances in scientific disciplines, technologies, and risk assessment practices can 

shift expectations about the data and information appropriate to establish reasonable certainty 

of no harm. Ultimately, we were led to ask:

Given the current state of our knowledge about the infant immune 

system and the known and anticipated properties of both bOPN and 

hOPN, is the existing data and prior experience with bOPN and hOPN: 

x generally recognized at this time 

x by experts qualified by scientific training and experience5

x as adequately demonstrating the safety of the intended use?

It became clear that the fundamental issue was whether the currently available data and

information presented by Arla were generally recognized as appropriate and sufficient, or 

whether a more robust discussion among qualified experts would identify substantive 

disagreement about the adequacy of the data, indicating lack of consensus and a potential need 

for additional data and information. In the case of OPN, given what is known about its MoAs 

and the intended infant population, we conclude that there is a strong need for experts to

4 Institute of Medicine (2004), “Strengthening the Current Process to Evaluate New Ingredients for Infant 

Formula” in Infant Formula: Evaluating the Safety of New Ingredients (Washington, DC: The National 

Academies Press), pp. 55-69. 

5 In this case, including experts in pediatric allergy and immunology.
Page 4 - Administrative File, GRN 000716

publicly debate the relevance and adequacy of the existing body of evidence for a safety 

determination. In other words, we have identified many questions potentially relevant to the 

possibility of immediate or long-term perturbation in the developmental trajectory of infant 

organ systems such as the immune system. Our preliminary consultations with FDA colleagues 

with relevant expertise, as noted above, suggest that these questions are reasonable given the 

available information and intended use. However, we have not seen any evidence that these 

questions have been considered and addressed from an appropriately diverse set of viewpoints 

among the scientific community of qualified experts. 

For GRN 000716, the notifier chose to provide evidence of general recognition by citing a

GRAS panel’s views on the data and information in the notice. The panel considered that this 

data and information was adequate to show safety. However, the panel’s conclusions do not 

provide, in our view, compelling evidence of general recognition by qualified experts. We have 

identified two specific issues that limit the panel’s ability to generate this evidence. These 

issues collectively undermine the panel’s ability to serve as an effective proxy for the views of 

the broader scientific community.

First, there is no evidence in the notice that the GRAS panel’s deliberations included the 

questions we describe in the GRN 000716 science memo. In our view, deliberations about the 

safety of the intended use did not include sufficiently detailed consideration of the known 

immunomodulatory properties of bOPN and their potential significance for this intended 

population. For this reason, we concluded that these deliberations were incomplete and of 

limited use in inferring consensus views of the totality of the evidence. Second, the panel 

appears to lack members with specialized expertise related to the known properties of bOPN 

and the intended population. Without the contributions and insights of this expertise to the 

panel’s deliberations, conclusions on the adequacy of the safety data are unlikely to be 

representative of views of relevant expert communities. 

It may be possible to address the issues discussed above, although there is no guarantee that 

efforts to elicit evidence of general recognition will be successful at this time. For example, 

even a well-constructed GRAS panel with appropriate and representative expertise to consider 

a particular intended use could ultimately conclude that there is not consensus among their 

peers about the usefulness and adequacy of the data available to support that use. Given the 

nature of the questions we have raised, generating convincing evidence that the available data 

is generally recognized as appropriate and sufficient may require sustained, substantial effort.

We do not expect that our questions could be resolved without evidence of a robust and 

expansive discussion of these issues. However, any sources of potential bias6 present would 

need to be identified and adequately addressed. Both the transparency of the discussion as well 

as the credibility of the discussants with respect to expertise and sources of potential bias are 

critical factors in any approach to generate compelling evidence of general recognition. The 

more open and inclusive the discussion (for example, a public symposium at a professional 

meeting with appropriate expertise in attendance, compared to a GRAS panel), the easier it

would be to make the case that issues of bias, expertise, and representation of viewpoints had 

6 Although the guidance “Best Practices in Convening GRAS Panels” (Ref. 1) is currently in draft, it may be helpful 

in illustrating the concept of sources of potential bias and some potential strategies for addressing them.
Page 5 - Administrative File, GRN 000716

been adequately addressed.

 _________________________

 Jeremiah Fasano

Reference:

1. FDA, “Draft Guidance for Industry: Best Practices for Convening a GRAS Panel,” 

(http://www.regulations.gov in Docket No. FDA-2017-D-0085), 2017. 

Jeremiah 

M. Fasano 

-S

Digitally signed 

by Jeremiah M. 

Fasano -S 

Date: 2019.03.25 

12:46:25 -04'00'
Page 6 - Administrative File, GRN 000716

cc: GRN 000716

E-mail cc: HFS-255 (SCarlson, RChanderbahn, MJDiNovi, RIMerker, SWestBarnette, 

PMGaynor, RShah, KKaneko, , RBonnette, LShepherd) HFS-850 (ALotze, CAssar)

R/D:HFS-255:Jeremiah Fasano:02/27/2018

Comments:HFS-255:KKaneko:02/28/2018

Edit:HFS-255:JMFasano:03/29/2018

Comment/Edit:HFS-255:PGaynor:04/16/2018 

Edit/Init:HFS-255:SCarlson:06/19/18

Edit:HFS-255JMFasano:07/05/2018

Comments:HFS-255:KKaneko:07/05/2018

Init:HFS-255:NBewry:07/11/2018 

Edit:HFS:255:JMFasano:03/10/2019

Comments:HFS-255:RIMerker:03/11/2019 

Comments:HFS-255:SChoudhuri:03/11/2019

Comments:HFS-255:PGaynor:03/13/2019

Comments:HFS-850:ALotze:03/13/2019

Comments:HFS-255:SCarlson:03/18/2019

F/T:HFS-255:JMFasano:03/21/2019
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:

• Why the safety factor of 50, instead of 100, is appropriate.

• 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:

• 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. 

• 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.