Workshop report: A study roadmap to evaluate the safety of recombinant human lactoferrin expressed in Komagataella phaffii intended as an ingredient in conventional foods - Recommendations of a scientific expert panel.

Human milk lactoferrin (hmLF) is a glycoprotein with well-known effects on immune function. Helaina Inc. has used a glycoengineered yeast, Komatagaella phaffii, to produce recombinant human lactoferrin (Helaina rhLF, Effera™) that is structurally similar to hmLF with intended uses as a food ingredient. However, earlier FDA reviews of rhLF were withdrawn due to insufficient safety data and unanswered safety questions the experts and FDA raised about the immunogenicity/immunotoxicity risks of orally ingested rhLF. Helaina organized a panel of leading scientists to build and vet a safety study roadmap containing the studies and safety endpoints needed to address these questions. Panelists participated in a one-day virtual workshop in June 2023 and ensuing discussions through July 2023. Relevant workshop topics included physicochemical properties of LF, regulatory history of bovine LF and rhLF as food ingredients in the FDA's generally recognized as safe (GRAS) program, and synopses of publicly available studies on the immunogenicity/alloimmunization, immunotoxicology, iron homeostasis, and absorption, distribution, metabolism, and excretion of rhLF. Panelists concluded that the safety study roadmap addresses the unanswered safety questions and the intended safe use of rhLF as a food ingredient for adults and agreed on broad applications of the roadmap to assess the safety and support GRAS of other recombinant milk proteins with immunomodulatory functions.

A review of the safety evidence on recombinant human lactoferrin for use as a food ingredient.

Published studies on the glycosylation, absorption, distribution, metabolism, excretion, and safety outcomes of orally ingested recombinant human lactoferrin (rhLF) were reviewed in the context of unanswered safety questions, including alloimmunization, allergenicity, and immunotoxicity potential of rhLF during repeated exposure. The primary objective was to summarize current safety data of rhLF produced in transgenic host expression systems. Overall, results from animal and human studies showed that rhLF was well tolerated and safe. Animal data showed no significant toxicity-related outcomes among any safety or tolerability endpoints. The no observed adverse effect levels (NOAEL) were at the highest level tested in both iron-desaturated and -saturated forms of rhLF. Although one study reported outcomes of rhLF on immune parameters, no animal studies directly assessed immunogenicity or immunotoxicity from a safety perspective. Data from human studies were primarily reported as adverse events (AE). They showed no or fewer rhLF-related AE compared to control and no evidence of toxicity, dose-limiting toxicities, or changes in iron status in various subpopulations. However, no human studies evaluated the immunomodulatory potential of rhLF as a measure of safety. Following this review, a roadmap outlining preclinical and clinical studies with relevant safety endpoints was developed to address the unanswered safety questions.

Perceived Palliative Care Education Needs of GP Trainees: A National Study.

CONTEXT: GP's provide palliative care in the community. Managing complex palliative needs can be challenging for GPs and even more so for GP trainees. During the postgraduate training period GP trainees are working in the community but have more time for education. This time in their career could present a good opportunity for palliative care education. For any education to be effective their educational needs must first be clarified. OBJECTIVES: To explore the perceived palliative care education needs and preferred education methods of GP trainees. METHODS: A national, multisite qualitative study involving a series of semistructured focus group interviews with third and fourth year GP trainees. Data were coded and analyzed using Reflexive Thematic Analysis. RESULTS: Perceived Educational Needs: five themes were conceptualized: 1) Empowerment vs. Disempowerment; 2) Community Practice; 3) Intra- and Interpersonal Skills; 4) Formative Experiences; 5) Contextual Challenges. PREFERRED EDUCATIONAL METHODS: Three themes were conceptualized: 1) Oriented Learning-experiential vs. didactic; 2) Practicalities; 3) Communication Skills. CONCLUSION: This is the first multisite national qualitative study exploring the perceived palliative care educational needs and preferred educational methods of GP trainees. Trainees expressed a unanimous need for experiential palliative care education. Trainees also identified means to address their educational requirements. This study suggests that a collaborative approach between specialist palliative care and general practice is necessary to create educational opportunities.

Weighted analysis of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose concentrations in human milk.

Over 150 human milk oligosaccharides (HMOs) have been identified and their concentrations in human milk vary depending on Secretor and Lewis blood group status, environmental and geographical factors, lactation stage, gestational period, and maternal health. Quantitation of HMOs in human milk has been the focus of numerous studies, however, comprehensive and weighted statistical analyses of their levels in human milk are lacking. Therefore, weighted means, standard deviations, medians, interquartile ranges, and 90th percentiles for 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) were calculated using random sampling and the levels of these HMOs in human milk reported in the literature. Probability distributions of the reported levels were also constructed. Although the levels reported in the published studies varied, the weighted means for 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL were calculated to be 2.58, 0.57, 0.94, 0.28, and 0.39 g/L, respectively, which are consistent with those that have been previously determined in other systematic analyses. Likely due to the use of weighting, the 90th percentiles were greater than the 95% confidence limits that have been previously calculated. Our study therefore provides accurate and important statistical data to help support the level of appropriate HMO supplementation in infant formula.

Safety and Metabolism of Long-term Administration of NIAGEN (Nicotinamide Riboside Chloride) in a Randomized, Double-Blind, Placebo-controlled Clinical Trial of Healthy Overweight Adults.

Nicotinamide riboside (NR) is a newly discovered nicotinamide adenine dinucleotide (NAD+) precursor vitamin. A crystal form of NR chloride termed NIAGEN is generally recognized as safe (GRAS) for use in foods and the subject of two New Dietary Ingredient Notifications for use in dietary supplements. To evaluate the kinetics and dose-dependency of NR oral availability and safety in overweight, but otherwise healthy men and women, an 8-week randomized, double-blind, placebo-controlled clinical trial was conducted. Consumption of 100, 300 and 1000 mg NR dose-dependently and significantly increased whole blood NAD+ (i.e., 22%, 51% and 142%) and other NAD+ metabolites within 2 weeks. The increases were maintained throughout the remainder of the study. There were no reports of flushing and no significant differences in adverse events between the NR and placebo-treated groups or between groups at different NR doses. NR also did not elevate low density lipoprotein cholesterol or dysregulate 1-carbon metabolism. Together these data support the development of a tolerable upper intake limit for NR based on human data.

Investigating the healthcare utilisation and other support needs of people with young-onset dementia.

INTRODUCTION: There exists a knowledge gap about the specific needs and utilisation of healthcare services by people with young-onset dementia (YOD), defined as being diagnosed before the patient is 65 years of age. Palliative care for dementia has received increasing attention, yet those with YOD have been overlooked. OBJECTIVE: To explore healthcare utilisation, including at end-of-life, of people with YOD in Ireland, using hospital electronic records for case finding. METHOD: We obtained Hospital In-Patient Enquiry data identifying all people with YOD admitted to three large urban hospitals between 2009 and 2016, and conducted a retrospective chart review. Information collected included demographics, medical and psychosocial history, functional capacity, last hospital admission, mortality and details regarding an advance care plan (ACP), using a standardised extraction form. RESULTS: Of the 121 patients identified, 50% were male. The commonest dementia types were: dementia secondary to Down's syndrome (16%), vascular (14%), frontotemporal (13%) and Alzheimer's disease (13%). 88% had ≥1 comorbid disease, including neurological (55%), cardiovascular (36%), and mental health illnesses (29%). Although 70% of people with YOD had an indication for an ACP (i.e. one or more markers of limited life expectancy), only 11% had any ACP recorded. 37% of patients had died, most commonly due to a complication of advanced dementia (e.g. aspiration pneumonia) rather than comorbid illness. CONCLUSION: People with YOD most commonly die from complications of dementia, as opposed to people with late-onset dementia, who often die with dementia. Advanced care planning appears to be suboptimal in people with YOD. More research is essential to inform future policies and services for this often neglected population.

Red meat and colon cancer: A review of mechanistic evidence for heme in the context of risk assessment methodology.

On October 26, 2015, IARC published a summary of their findings regarding the association of cancer with consumption of red meat or processed meat (IARC 2015; The Lancet Oncology 2015). The Working Group concluded that there is limited evidence in human beings for carcinogenicity from the consumption of red meat and inadequate evidence in experimental animals for the carcinogenicity of consumption of red meat. Nevertheless, the working group concluded that there is strong mechanistic evidence by which ingestion of red meat can be linked to human colorectal cancer and assigned red meat to Group 2A "probably carcinogenic to humans". The Working Group cited supporting mechanistic evidence for multiple meat components, including those formed from meat processing, such as N-nitroso compounds (NOC) and heterocyclic aromatic amines, and the endogenous compound, heme iron. The mechanism of action for each of these components is different and so it is critical to evaluate the evidence for each component separately. Consequently, this review critically examined studies that investigated mechanistic evidence associated with heme iron to assess the weight of the evidence associating exposure to red meat with colorectal cancer. The evidence from in vitro studies utilized conditions that are not necessarily relevant for a normal dietary intake and thus do not provide sufficient evidence that heme exposure from typical red meat consumption would increase the risk of colon cancer. Animal studies utilized models that tested promotion of preneoplastic conditions utilizing diets low in calcium, high in fat combined with exaggerations of heme exposure that in many instances represented intakes that were orders of magnitude above normal dietary consumption of red meat. Finally, clinical evidence suggests that the type of NOC found after ingestion of red meat in humans consists mainly of nitrosyl iron and nitrosothiols, products that have profoundly different chemistries from certain N-nitroso species which have been shown to be tumorigenic through the formation of DNA adducts. In conclusion, the methodologies employed in current studies of heme have not provided sufficient documentation that the mechanisms studied would contribute to an increased risk of promotion of preneoplasia or colon cancer at usual dietary intakes of red meat in the context of a normal diet.

The relevance of international assessments to GRAS determinations.

A discussion of the risk assessment process as applied to the Generally Recognized As Safe (GRAS) determination of safety for new ingredients can benefit from an international perspective. When we think about how risk assessments are performed around the world it is critical to assess what can be learned. What are the similarities? What are the differences? What are the takeaways? It is important to talk about the similarities in processes, because it validates the approach taken by risk assessors who are charged with protecting the food supply. It is also instructive to evaluate the differences in order to determine where improvements can be made to our process.

An innovative approach to the safety evaluation of natural products: cranberry (Vaccinium macrocarpon Aiton) leaf aqueous extract as a case study.

Assessment of safety for a food or dietary ingredient requires determination of a safe level of ingestion compared to the estimated daily intake from its proposed uses. The nature of the assessment may require the use of different approaches, determined on a case-by-case basis. Natural products are chemically complex and challenging to characterize for the purpose of carrying out a safety evaluation. For example, a botanical extract contains numerous compounds, many of which vary across batches due to changes in environmental conditions and handling. Key components integral to the safety evaluation must be identified and their variability established to assure that specifications are representative of a commercial product over time and protective of the consumer; one can then extrapolate the results of safety studies on a single batch of product to other batches that are produced under similar conditions. Safety of a well-characterized extract may be established based on the safety of its various components. When sufficient information is available from the public literature, additional toxicology testing is not necessary for a safety determination on the food or dietary ingredient. This approach is demonstrated in a case study of an aqueous extract of cranberry (Vaccinium macrocarpon Aiton) leaves.

Assessment of the potential allergenicity of a Milk Basic Protein fraction.

BACKGROUND: A specific basic fraction of bovine milk, termed Milk Basic Protein (MBP), has the potential to provide nutritionally important benefits if used as a food ingredient. Although derived from milk, MBP is intended for use as an ingredient in other foods. Cows' milk is a well studied, commonly allergenic food. Although the proteins in MBP are not identified as milk allergens, food products containing MBP will be labelled as containing milk as a caution to milk allergic consumers under food labelling guidelines in the US and the European Union as MBP has not been demonstrated to be free of milk allergens. However, as part of an overall safety evaluation of MBP, the developers sought to evaluate the potential allergenicity of the primary protein components for characteristics of allergenic food proteins and to assess whether intake of these proteins at intended use levels could present a significant new allergenic risk for consumers. OBJECTIVE: To evaluate the potential allergenicity of the five identified proteins in MBP. While extensive studies have not demonstrated allergenicity of lactoferrin, the four other proteins are less studied. The four were tested here by sequence identity comparison to known allergens, and for stability of these proteins in acidic pepsin as a characteristic common to many food allergens. METHODS: Sequences of the proteins were compared to those listed in AllergenOnline.com, by methods recommended for the evaluation of proteins introduced in crops through genetic engineering. Pepsin stability was assessed by incubating the various proteins in simulated gastric fluid at pH 1.2 with porcine pepsin for up to 60 min at 37 degrees C, with samples withdrawn and analyzed at specific times. RESULTS: No significant sequence similarities were identified for the MBP proteins compared to known allergens. All but one of the protein components of MBP were digested relatively quickly by pepsin. The more stable protein will be of low abundance as consumed in contrast to most pepsin-stable food allergens. CONCLUSIONS: Based on molecular characteristics and expected exposure, the protein components in MBP are unlikely to present any increased risk of allergy for milk allergic subjects or of cross-reactivity for other allergic subjects. However, since the proteins are derived from milk, products containing MBP will need to be labelled as containing milk proteins to warn milk allergic subjects of the potential risk of allergic reactions.

Safety determination for the use of bovine milk-derived lactoferrin as a component of an antimicrobial beef carcass spray.

Bovine milk-derived lactoferrin (BMDL), an iron-binding glycoprotein, is known to be an effective natural antimicrobial. It is used as a spray, applied electrostatically, to raw beef carcasses to detach bacteria adhering to the surface in order to reduce microbial contamination. The use of BMDL as a component (at not more than 2% by weight) of an antimicrobial spray was determined Generally Recognized As Safe (GRAS) (GRN 67) for three proposed uses (i.e., beef carcasses, subprimals, and finished cuts), provided that the ingredient statement of food products that contain milk-derived lactoferrin identifies the source of the protein. The use of BMDL spray on only beef carcasses (not subprimals or finished cuts) at a level not to exceed 0.20 ml of formulation per kg of beef was determined safe without the requirement of labeling of food products so treated. The two key components of the assessment are: (1) a determination that exogenous lactoferrin exposure (resulting from its application to beef carcasses) is in the range of existing background exposures of lactoferrin as a result of lactoferrin found naturally in beef, and (2) a determination that this potentially small incremental increase in lactoferrin is safe (i.e., there is no reasonable expectation that BMDL will become an allergen under the conditions of its intended use).

Safety evaluation of sources of docosahexaenoic acid and arachidonic acid for use in infant formulas in newborn piglets.

Human milk provides small quantities of preformed docosahexaenoic acid (DHA) and arachidonic acid (ARA), usually less than 1% of total fatty acids. Vegetable oil blends commonly used in infant formulas have, until recently, provided the essential fatty acid precursors for these long-chain polyunsaturated fatty acids (LCPUFA), but no preformed DHA and ARA. This study evaluated the safety of ingredient sources of DHA and ARA for use in infant formulas in a neonatal piglet model. Newborn piglets were allowed to suckle for 3 days and then divided into 4 feeding groups of 6 males and 6 females. Piglets were bottle-fed at frequent feeding intervals until 19 days of age. The composition of the piglet formulas was modeled after standard milk-based formulas for human infants while meeting nutritional requirements for piglets. Formulas were a control formula (no added DHA or ARA), a DHA formula providing 55 mg DHA/100 Cal, an ARA formula providing 96 mg/100 Cal ARA, and a DHA+ARA formula providing 34 mg DHA and 62 mg ARA/100 Cal. All formulas were equal in fat content and provided approximately 1000 Cal/l. The ARA-rich oil was from a fermentation product of Mortierella alpina (40 wt.% fatty acids as ARA) and DHA was from high DHA tuna oil (25 wt.% fatty acids as DHA). There were no test article related effects of DHA and/or ARA indicative of an adverse health consequence to the animals seen in the clinical signs, body weights, food consumption, clinical chemistry, hematology, organ weights or gross or histopathology. The findings in this neonatal animal study support the safety of these ingredient oil sources of DHA and ARA for use in infant formulas.