Dominique Turck

Jean-Louis Bresson

Barbara Burlingame

Tara Dean

Susan Fairweather-Tait

Marina Heinonen

Karen Ildico

Inge Mangelsdorf

Harry J Mcardle

Androniki Naska

Monika Neuh € Auser-Berthold

Gra _ Zyna Nowicka

Kristina Pentieva

Yolanda Sanz

Alfonso Siani

Anders Sj

Martin Stern

Daniel Tom

Marco Vinceti

Peter Willatts

Karl-Heinz Engel

Rosangela Marchelli

Morten Poulsen

Josef Rudolf Schlatter

Mathias Amundsen

Henk Van Loveren

D Bresson

Mangelsdorf I Hirsch-Ernst Ki

Mcardle Hj

Neuh € Auser-Berthold

Safety of Whey basic protein isolates as a novel food pursuant to Regulation (EU) 2015/2283 EFSA Panel on Dietetic Products, Nutrition and Allergies (EFSA NDA Panel)

Keywords: basic whey protein isolate, novel food, ingredient, safety

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on whey basic protein isolate as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is obtained by ion exchange chromatography of skimmed cow's milk. The applicant intends to market the NF in infant and follow-on formulae and meal replacement beverages, dietary foods for special medical purposes and as food supplements. The highest estimated intake of the NF based on the proposed uses and use levels would be 24.8 mg/kg body weight (bw) per day in infants and 27.8 in toddlers. The information provided on composition, specifications, production process and stability of the NF do not raise safety concerns. Taking into account the composition of the NF and the intended use levels, the Panel considers that the consumption of the NF is not nutritionally disadvantageous. The Panel considers that there is no concern with respect to genotoxicity. The no observed adverse effect level (NOAEL) of a subchronic 13-week rat study was 2000 mg/kg bw per day. Considering the source, the production process and nature of the NF, the Panel considers the margin of exposure (MOE) of 154 to be sufficient for the adult population (on a high-estimated intake of 13 mg/kg bw). For infants and toddlers, the MOE would be at least 81 and 72, respectively. Taking into account the composition of the NF, its source, the history of consumption of the main components of the NF, the production process and that the NOAEL in a subchronic rat study was the highest dose tested the Panel considers that also the MOE for infants and toddlers are sufficient. The Panel concludes that the novel food ingredient, whey basic protein isolate, is safe under the proposed uses and use levels.

Table of contents

Background and Terms of Reference as provided by the European Commission

On 22nd August 2016, the company S.A.S. submitted a request in accordance with Article 4 of the Novel Food Regulation (EU) 258/19971 to place on the market whey basic protein isolate as a novel food (ingredient) (NF).

On 27 June 2017, the Food Safety Authority of Ireland forwarded to the Commission its initial assessment report, which came to the conclusion that the whey protein isolate meets the criteria for acceptance of a novel food defined in Article (3)1 of the regulation (EC) No 258/97.

On 4 July 2017, the Commission forwarded the initial assessment report to the other Member States. Several Member States raised objections or submitted comments.

The concerns of a scientific nature raised by the MS can be summarised as follows:

• Qualitative and quantitative data on the proteins contained in the NF were requested to understand the profile of the food.

• Proof of accreditation of the laboratories used to perform the relevant tests was requested.

• The description of the method of determination for bovine lactoferrin (bLF) and lactoperoxidase (bLP) was requested.

• Analytical results for content of arsenic in the NF were requested.

• The use of gluten or wheat was used as a processing aid in spray drying to prevent clumping.

• The enzymatic activity of bLP, and the potential effect that the NF may have on the food matrixes to which it will be added. As this may affect the storage stability.

• Additional data on the storage stability of the product in other packaging and intended areas of application was requested.

• A safety study involving infant and follow on formula was requested.

• The toxicological relevance of the results shown in a 6-week developmental toxicology study in juvenile rats. The effect of the NF on body weight and increased thymus weight.

According to Article 35 (1) of Regulation (EU) 2015/2283 2 , any request for placing a novel food on the market within the Union submitted to a Member State in accordance with Article 4 of Regulation (EU) 258/1997 and for which the final decision has not been taken before 1 January 2018 shall be treated as an application under this Regulation.

In accordance with Article 10 (3) of Regulation (EU) 2015/2283, EFSA shall give its opinion as to whether the update of the Union List referred to in Article 10 (1) is liable to have an effect on human health.

2.

Data and methodologies

Data

The safety assessment of this NF is based on data supplied in the application and information submitted by the applicant following EFSA request for supplementary information. During the assessment, the Panel identified additional data which were not included (US FDA, 2017).

Administrative A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of a NF application. 4 As indicated in this guidance, it is the duty of the applicant to provide all of the available (proprietary, confidential and published) scientific data, including both data in favour and not in favour to supporting the safety of the proposed NF.

This NF application includes a request for protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283. Data claimed to be proprietary by the applicant include: Armor Prot eines (2013; unpublished), Armor Prot eines (2017; unpublished), [START_REF] Schmitt | Rapport Provisoir de L'Etude Sur L'Effet de la Consommation de Lactochol ® Sur Les Parametres de L'Atherosclerose[END_REF]unpublished), [START_REF] Silvano | 13-week toxicity study by the oral route (gavage) in rats followed by a 4-week treatment-free period[END_REF]unpublished), Sire (2012a;unpublished, 2012b;unpublished) and Sp ezia (2013; unpublished).

Methodologies

The assessment follows the methodology set out in the EFSA guidance on NF applications and the principles described in the relevant existing guidance documents from the EFSA Scientific Committee. The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 and in Article 7 of the Commission Implementing Regulation (EU) 2017/2469. This assessment concerns only risk that might be associated with consumption of the NF under the proposed conditions of use, and is not an assessment of the efficacy of basic whey protein isolate with regard to any claimed benefit.

Assessment

Introduction

The NF is a whey basic protein isolate obtained from skimmed cow's milk by chromatographic fractionation and has a protein content higher than 90%. The applicant intends to market the NF as an ingredient for infant and follow-on formulae, meal replacement beverages, dietary foods for special medical purposes and food supplements.

Identity of the NF

The whey basic protein isolate is produced from bovine skimmed milk. The protein fraction of the NF is composed mainly of two minor whey basic proteins, bLF and bLP which account for approximately 80% of the NF and contains only low quantities of the predominant proteins of bovine whey (i.e. b-lactoglobulin and a-lactalbumin). bLF and bLP constitute only 2-4% of the whey proteins from cow's milk.

Production process

The production of whey basic proteins starts with removal of fat by centrifugation of bovine milk. The whey basic protein fraction is isolated by ion-exchange chromatography using a cation resin. From the resin, the basic whey proteins are eluted with a NaCl solution, which again is largely removed by ultrafiltration. For reduction of the microbial load, the product is microfiltrated and pasteurised, before it is spray-dried. Following a question from a Member State (MS), the applicant clarified that no gluten or wheat products were used as a processing aid in spray-drying to prevent clumping. The NF is manufactured in accordance with good manufacturing practice (GMP) and the hazard analysis critical control point (HACCP) principles in a production plant certified under ISO 22000. The techniques used in the production process of the NF are commonly used in the dairy industry and are comparable to techniques used in production of bLF previously assessed by (EFSA NDA Panel, 2012a,b).

The Panel considers that the production process is sufficiently described and does not raise safety concerns.

Compositional data

The applicant provided batch-to-batch analyses of five different production lots (Table 1). According to the analyses, the NF has a protein content higher than 90%. bLF (25-75%) and bLP (10-40%) are the two main constituents of the NF. Following a question from one MS on other contained proteins, data were provided on the 10 most abundant proteins: secretory component < 5%, complement C3 < 2%, b-lactoglobulin < 2%, aS1-Casein < 2%, lactophorin < 2%, pancreatic ribonuclease (RNase-4) < 2%, tetranectin (CLEC3B) < 1% and b-2-glycoprotein 1 (apolipoprotein H) < 1%. Two proteins found in lower quantities in the NF are transforming growth factor (TGF-b2; 0.012-0.018%) and insulin-like growth factor (IGF-1; 0.0015%).

The content of moisture, lactose and fat was below 5.5, 3.0, and 4.5%, respectively.

The Panel considers that the information provided on the composition of the NF is sufficient and does not raise safety concerns.

Stability

In the dossier, the applicant provided a summary of stability studies of the NF in a double ethylene bag packed in a cardboard box under normal storage conditions (20°C and relative humidity 40-50%). The studies showed that no significant microbial growth in the NF and the total protein and TGF-b2 contents did not show pronounced changes over time. However, the moisture content increased by 6% after 12-18 months in one of the stability studies and after 27 months in one batch of the other study. The applicant, therefore, suggested limiting the use by date of the product to 12 months. In response to a comment from a MS, a stability study with dietary foods for special medical purposes was provided by the applicant. The product was supplemented with the NF to a content of TGF-b2 of between 77 and 118 lg/100 g after formulation. The samples were stored under ambient conditions up to 18 months. After storage for 12 months, the TGF-b2 content had decreased by 10% of the initial TGF-b2 content.

Responding to comments from two MS regarding the enzymatic activity and the potential effect of peroxidases to form free radicals in the NF, the panel agreed with the applicant's response that such reaction does not occur in the NF since both thiocyanate ion and hydrogen peroxide are required to exert its peroxidase activity (FAO, 2002).

The Panel considers that the data provided are sufficient with respect to the stability of the NF.

Specifications

The specifications as proposed by the applicant are presented in Table 2. The analyses were performed using validated methods. The results from batch-to-batch analyses provided in Section 3.4. were within the specification limits and contaminants were below the limits set for both the microbiological and chemical contaminants.

The Panel considers that the information provided on the specifications of the NF is sufficient and does not raise safety concerns.

3.6.

History of use of the NF and/or of its source 3.6.1. History of use of the source Bovine whey, whey protein concentrates and demineralised whey powder are consumed in the EU. Whey protein concentrates produced with a range of techniques including ion exchange resins were approved as a food ingredient in the USA in 1981 [START_REF] Us Fda | GRAS status of whey, whey products and hydrogen peroxide; Final rule (21 CFR Part 184)[END_REF]. The per capita consumption of whey was estimated at 1.6 lbs (dry matter)/year for 2013, equivalent to approximately 2 grams (dry matter)/person per day in the USA (USDA-ERS, 2016).

History of use of the NF

Some whey basic protein isolates have been granted GRAS status and are permitted to be marketed in the United States (US FDA, 2000, 2006). Also, several bLF products have received GRAS status (US FDA, 2001a,b, 2003, 2014a,b, 2017). According to the applicant, bLF is permitted for use in Japan, China, Korea and Taiwan for both infant formulae and conventional foods, and in infant and follow-on formulae since 1986 in Japan.

In 2012, EFSA published two opinions on the safety of bLF (EFSA, 2012a,b). In these opinions, the safety of bLF was assessed for addition to several food categories (i.e. infant and follow-on formulae, dietetic food for special medical purposes, sports nutrition, non-alcoholic beverages, cakes and pastries, products derived from cheese, milk-based products, cold snacks and sweets). The proposed maximum use of reconstituted (ready-to-drink) infant formulae and follow-on formulae was of 100 mg bLF/100 mL. The Panel considered that the estimated maximum daily intake of 3.4 g bLF for adults, and for toddlers, the 95th percentile intake of 203 mg/kg bw per day were safe.

EFSA has not previously evaluated the safety of bLP. Bovine lactoperoxidase is accepted in dietary supplements (1-2 mg/serving) in the USA (NIH, 2016), and in Japan as food additive (MHLW, 2014) and food supplements. In addition, bLP-containing systems have been assessed by JECFA (1990) and accepted in New Zealand (FSANZ, 2002) as processing aids.

Table 3 provides information on the concentrations of lactoferrin, lactoperoxidase and TGF-b2 found in human and bovine milk and infant formulae. Human milk 100-320 (a) 0.077 (b) 0.04-1.9 (mean % 0.53) (c) 0.05-1.3 (geometric mean = 0.27) (d) 0.2-1.5 (mean = 0.6)

Cow's milk 10-15 (e) 2.4-3.4 (f) 6.6 (g) 1.3-7.1 (h) Infant formulae 12.7-19.1 (i) 3.0-4.5 (i) 0.28-1 (j) The applicant intends to target infants and toddlers (infant and follow-on formulae), the general adult population (meal replacement beverages) and the general population (foods for special medical purposes and food supplements).

Proposed uses and use levels

The applicant intends to market the NF as an ingredient for the food categories presented in Table 4. The rationale for the use level in infant and follow-on formulae given by the applicant is to achieve intake levels of TGF-b2 comparable to those of breast-fed infants.

Table 5 provides the calculated maximum use levels for bLF, bLP and TGF-b2 by considering their specification upper limits and the proposed maximum use levels of the NF for the intended food categories.

The Panel notes that the use levels for bLF in two NF applications assessed and considered safe by the Panel were up to 100 mg/100 g in reconstituted (ready-to-drink) infant formulae, which is about 30 times higher than the bLF concentration (i.e. 2.9 mg/100 mL, Table 5), potentially resulting from the addition of the proposed use level of the NF. In 2012, the Panel considered bLF as safe in infant formula at the proposed use level in infant formula at 1 g/L (EFSA NDA Panel, 2012a).

The applicant reports that analytical data on the occurrence of bLP in infant formula could not be retrieved, but provided an estimate (i.e. 3.0-4.5 mg/100 mL, Table 3). Considering the proposed use level of the NF for reconstituted infant formula (i.e. 3.9 mg/100 mL) and the specification upper limit 3.9 mg/100 mL (b) Follow-on formula N/a 30 mg/100 g (a) 4.2 mg/100 mL for bLP in the NF (40%), the addition of the NF to infant formulae could result to an increase of about 35% (4.5 mg/100 mL + 1.6 mg/100 mL).

The addition of the NF could result in a maximum increase of 0.7 lg/100 mL of bovine TGF-b2 present in bovine milk-based infant formulae at reported concentrations ranging from 0.28 to 1 lg/ 100 mL according to [START_REF] Jouni | Concentration of transforming growth factor-beta in an infant formula from five countries and human milk from Mexico and the US[END_REF]. This increase could result in a total concentration of TGF-b2 in infant formula at the higher range reported for the TGF-b2 in human breast milk (up to 1.9 lg/ 100 mL; Table 3).

Anticipated intake of the NF Infant and follow-on formulae

Proposed use levels of the NF through infant and follow-on formulae could lead to a consumption of the NF of 20 and 23 mg/kg bw per day in infants and toddlers, respectively (Table 6).

Meal replacement beverages

The applicant provided intake estimates from meal replacement beverages in the UK National Diet and Nutrition Survey (UKDA, 2014). The number of consumers of meal replacement beverages was low, so only the all-user data are presented (Table 7). Under the proposed conditions of use, the intake of meal replacement beverages is highest in children between the age of 4 and 10 where they can account for up to 3.9 mg/kg bw per day. In the adult population, they can account for between 1 and 1.7 mg/kg bw per day. It is worth noting that the statistical reliability of the results is low due to the low number of consumers. In a scenario with the consumption of 3 meals per day, the intake of the NF in the adult population would be 300 mg/day (for a 70 kg adult) or 4.29 mg/kg bw per day. For children (4-10 years of age), a daily consumption of 300 mg of the NF would correspond to 13 mg/kg bw per day when considering a mean default body weight of 23 kg (EFSA Scientific Committee, 2012).

Table 6: Mean and 95th percentile intakes of the NF from infant and follow-on formulae, estimated on the basis of consumption data from Germany [START_REF] Kersting | Measured consumption of commercial infant food products in German infants: Results from the DONALD study (Dortmund Nutritional and Anthropometrical Longitudinally Designed)[END_REF]

Foods for special medical purposes

For the adult population, the maximum intended intake level (i.e 610 mg/day) proposed as food for special medical purposes would lead to an intake of 8.7 mg/kg bw per day, when considering a body weight of 70 kg.

For the younger population (specified by the applicant as children ≤ 3 years of age), the maximum intended intake levels (i.e. 58 mg per day) proposed as food for special medical purposes would lead to an intake of 4.8 mg/kg bw per day, when considering the mean body weight of 12 kg suggested by the EFSA Scientific Committee ( 2012) to be used in risk assessment for this age group.

Food supplements

The intended target population and use levels correspond to those as indicated for FSMPs.

Combined intake of the NF

The proposed uses and use levels could lead to intakes of the NF up to 24.8 mg/kg bw per day for infants (20 + 4.8 mg/kg bw from infant formula and FSMP or food supplements) and 27.8 mg/kg bw per day for toddlers (23 + 4.8 mg/kg bw from follow-on formula and FSMP or food supplements).

Adults may have an intake of up to 13 mg/kg bw per day, 8.7 mg/kg bw per day coming from food supplement use in addition to 4.3 mg/kg bw per day from meal replacements (when considering a default body weight of 70 kg).

Absorption, distribution, metabolism and excretion (ADME)

The applicant has not provided any studies regarding the metabolism of the NF. However, a limited number of studies on ADME of lactoferrin and lactoperoxidase were provided.

In humans, lactoferrin seems to be stable towards proteolytic activity and resistant to digestion in the stomach [START_REF] Troost | Gastric digestion of bovine lactoferrin in vivo in adults[END_REF]. In a human study, where adults (n = 6) were given bLF-radiolabelled iodine-123, about 31% of the initial activity was seen in the plasma within about 60 min [START_REF] Khan | Biodistribution of human lactoferrin in the gastrointestinal tract[END_REF]. Plasma levels rapidly decreased and about 5% of the initial dose was observed after 6 h. In breast-fed infants of 1 week of age, 2-6% of the lactoferrin from the breast milk was found in the faeces, which was decreased to less than 2% in 4-month-old infants [START_REF] Davidson | Persistence of human milk proteins in the breast-fed infant[END_REF]. It has been proposed that absorbed intact lactoferrin is either taken up by receptor-mediated endocytosis into phagocytic cells or by endocytic uptake by liver cells [START_REF] Levay | Lactoferrin: a general review[END_REF].

Only very limited information about the metabolic fate of bLP is available. It was reported that lactoperoxidase was inactivated in the presence of pepsin (pH 2.5) but not inactivated by the gastric juice of an infant (pH 5) [START_REF] Kussendrager | Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications[END_REF].

With regard to their suitability to assess human safety, rats were considered as a suitable species since adult rats have been shown to absorb native bLF [START_REF] Takeuchi | Evidence of lactoferrin transportation into blood circulation from intestine via lymphatic pathway in adult rats[END_REF]. Moreover, orally administered bLF was found in the liver, kidneys, gall bladder, spleen and brain of adult mice [START_REF] Fischer | Uptake of ingested bovine lactoferrin and its accumulation in adult mouse tissues[END_REF] detected using an enzyme-linked immunosorbent assay (ELISA). The Panel considers that the provided studies show absorption of bLF in the intestine of rats, and that rats therefore are suitable to study bLF toxicity.

Nutritional information

The proteins found in the NF are comparable to those found in the native cow's milk as little chemical alteration occurs in processing of the product. Many of these proteins have human equivalents of varying sequence homology. The homology between human and bLF, bLP and TGF-b2 is approximately 70% [START_REF] Wal | Bovine milk allergenicity[END_REF], 83% (Armor proteins, 2014) and 99% (according to BLAST analysis performed in 'NCBI, database Non-redundant protein sequences (nr)' by EFSA), respectively. The applicant proposes to add the NF as an ingredient in infant and follow-on formula, to achieve levels of TGF-b2 comparable to those resulting from consumption of human milk.

Lactoferrin is an iron-binding protein and lactoperoxidase is a haem-bound protein. Considering the specification limit for iron in the NF (≤ 25 mg/100 g; Table 2) as well as the intake estimates of the NF (13 mg/kg bw per day in adults and 24.8 mg/kg bw per day in infants) provided in Section 3.7.3, the intake levels could lead to an intake of iron of 3.3 lg/kg bw per day in adults, and 6.2 lg/kg bw per day in infants. These values represent less than 5% of the dietary reference value for iron established by the Panel, i.e. for infants 8 mg/day, children (aged 1-6 years) 5 mg/day, men 6 mg/day, premenopausal women 7 mg/day (EFSA NDA Panel, 2015).

The Panel considers that consumption of the NF is not nutritionally disadvantageous.

Toxicological information

The applicant provided two in vitro genotoxicity studies, one subchronic 90-day study and one 6-week developmental toxicity study conducted with the NF. Toxicological studies of two commercial whey protein isolates were also provided: 'Milk Basic Protein' (MBP) (54% bLF, 41% bLP) [START_REF] Kruger | Safety evaluation of a milk basic protein fraction[END_REF] and 'Lactermin' (bLF and bLP together > 50%, smaller amounts of IGF-I, IGF-II, PDGF, FGF, TGF-b and betacelluli) [START_REF] Dyer | In vitro and in vivo safety studies of a proprietary whey extract[END_REF] to substantiate the safety of the NF.

Genotoxicity

Two bacterial reverse mutation tests were performed in Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 using the preincubation method and the plate incorporation method, in compliance with OECD TG 471 [START_REF] Forster | Safety evaluation of a whey protein fraction containing a concentrated amount of naturally occurring TGF-b2[END_REF]. Six concentrations of the NF were used for each bacterial strain: from 156.3 to 5,000 lg/plate using three plates per concentration with and without the presence of a metabolic activation system (S9 mix). The NF did not induce an increase in the number of revertants, either with or without S9 mix, in any of the five strains.

The NF was also assessed in an in vitro mammalian cell micronucleus test with L5178Y TK+/À mouse lymphoma cells, in compliance with OECD 487 [START_REF] Forster | Safety evaluation of a whey protein fraction containing a concentrated amount of naturally occurring TGF-b2[END_REF]. Based on the results of the cytotoxicity measurements and the precipitation of the substance at high concentrations, doses for the main study were selected. The test was performed in two independent main experiments in the presence and absence of a metabolic activation system (S9 mix) using seven concentrations from 39.06 to 2,500 lg/mL. In the short-term exposure, cells were incubated for 3 h with and without metabolic activation and three treatments, i.e. 156.3, 312.5 and 625 lg/mL (experiments 1 and 2, with S9 mix and experiment 1, without S9 mix) were evaluated. In the long-term exposure, cells were exposed to the NF for 24 h in the absence of metabolic activation, and three treatments, i.e. 312.5, 625 and 1,250 lg/mL, were evaluated. Without metabolic activation, no significant increase in the frequency of micronucleated cells was noted after either 3-or 24-h treatment. In the short-term exposure with metabolic activation, the frequency of micronucleated cells was increased by 3.5-fold at 625 lg/mL, but this was not statistically significant and remained within historical control range. No increase was seen in the second experiment using the same study conditions. The Panel concludes that the test item did not induce chromosome aberrations or damage to the cell division apparatus.

Taking into account the nature, source and production process of the NF, the Panel considers that there are no concerns regarding genotoxicity. This was supported by the tests performed.

Subchronic toxicity

The applicant provided a subchronic (13 weeks) toxicity study performed with the NF in compliance with OECD TG 408 [START_REF] Forster | Safety evaluation of a whey protein fraction containing a concentrated amount of naturally occurring TGF-b2[END_REF]. Groups of 6-week-old Sprague-Dawley rats (16/sex per group in control and high-dose groups and 10/sex per group in mid-and low-dose groups) were administered the NF suspended in 0.9% NaCl by oral gavage at a dosage volume of 10 ml/kg. This corresponded to doses of 0, 600 (low), 1,200 (mid) or 2,000 (high) mg of the NF/kg bw per day. At the end of the treatment period, 10 animals in each group were sacrificed, except for the first six animals per sex of groups 1 and 4, which were kept for a 4-week treatment-free recovery period.

No statistically significant differences were seen in food consumption, body weight, clinical and ophthalmological examinations between control and test groups. One male in the high-dose group suffered from abscess in the lateral region of the thorax and was sacrificed prematurely at Day 33. This finding is most likely due to an injury caused by the gavage. Mean cell haemoglobin concentration was significantly lower in the high-dosed males (32.90 vs. 33.30 in control), but this was not accompanied by findings in related parameters in this group. A significantly higher mean reticulocyte percentage and lower mean thrombin time were seen in mid-dose males. In females, a lower mean cell haemoglobin was seen in the low-dose group. None of the above-mentioned haematologically effects are considered treatment related. In blood biochemistry, no significant differences were seen between high-dosed animals and controls. A few significant findings were seen in the lower dose groups, but the changes are judged as incidental. A decreased urine volume, increased specific gravity and decreased pH were observed in females administered with 2,000 mg/kg bw per day. The differences noted were small, were only observed in one sex and were considered not to be adverse. No differences in organ weights were attributed to treatment with the NF. In addition, no macroscopic or microscopic findings were seen. The Panel considers that the highest dose tested (i.e. 2,000 mg/kg bw per day) is the no observed adverse effect level (NOAEL) of this study.

The applicant also provided subchronic (13 weeks) toxicity studies of MBP [START_REF] Kruger | Safety evaluation of a milk basic protein fraction[END_REF] and Lactermin [START_REF] Dyer | In vitro and in vivo safety studies of a proprietary whey extract[END_REF]. In both studies, the study authors came to the conclusion that these protein isolates did not give an indication of toxicity. In [START_REF] Kruger | Safety evaluation of a milk basic protein fraction[END_REF], a significant lower body weight was seen in female rats given 2,000 MBP/kg bw per day (highest dose) between day 14 and day 63. At 90 days, this effect was no longer seen. No effect on body weight was seen in male rats.

Chronic toxicity and carcinogenicity

No studies were provided assessing chronic toxicity or carcinogenicity.

Reproductive and developmental toxicity

A 6-week developmental toxicity study was performed with the NF in compliance with the USFDA Guidance for Industry, Nonclinical Safety Evaluation of Paediatric Drug Products [START_REF] Forster | Safety evaluation of a whey protein fraction containing a concentrated amount of naturally occurring TGF-b2[END_REF]. The objective of the study was to evaluate the potential effects of the NF on the development of juvenile rats, following daily oral (gavage) administration from post-natal day (PND) 7 to PND 49 inclusive. Pregnant female Sprague-Dawley rats were received in the test facility on gestation day (GD) 14. Following parturition, the litters were culled on PND 4 to standardised litters of 2M + 2F or 3M + 3F. The test group of 16 rats/sex was given 600 mg/kg bw per day of the NF. The control group (16 rats/sex) received the vehicle (NaCl 0.9%) only. At the end of the treatment period, 10 animals in each group were sacrificed. The remaining six animals were kept for a 4-week treatment-free recovery period. Despite the fact that there is exposure to both the NF and maternal milk during the early post-natal period, the Panel notes that there is no information on the relative intake of protein from either source. In addition, only one dose of the NF and one control group were tested. The Panel notes that no conclusion can be drawn from this study.

The applicant also provided a developmental toxicity study on a related whey basic protein isolate (MBP) which was performed on healthy male and female Crj:CD (SD) IGS rats (in compliance with the 'Guidelines for Designation of Food Additives and for Revision of Standards for Use of Food Additives'', Notification No. 29 of the Environmental Health Bureau, Ministry of Health and Welfare, Japan, March 22, 1996[START_REF] Kruger | Safety evaluation of a milk basic protein fraction[END_REF]. The study utilised one treatment group and one control group; 20 pregnant females were included per group. MBP was administered daily by gavage at a dose of 2,000 mg/kg bw per day on Days 7-17 of gestation.

No adverse clinical effects were observed in the dams, and no differences between treated and control animals were seen for body weight, body weight gain, food consumption, number of corpora lutea, number of implantation sites, number of live and dead foetuses, number of resorbed embryos, viability indices of foetuses, sex ratio, placental weight and body weight of foetuses. In live foetuses, there were no external, visceral or skeletal anomalies or variations. These results show no adverse effects of MBP on reproduction or development in rats.

Human data

The applicant did not provide human studies performed with the NF. The applicant provided eight human studies with products containing main components of the NF. Six of these studies, however, investigated dose levels for bLF and bLP which were considerably below the proposed maximum intake levels of this NF. One open-label study tested a product without bLF in psoriasis patients (Armor Prot eines SAS., 2013; unpublished) and one randomised, double-blind, placebo-controlled trial with a product containing bLF, bLP and TGF-b2 in amounts complying with the specifications of the NF, studied the effects on markers of atherosclerosis in hypercholesterolemic subjects [START_REF] Schmitt | Rapport Provisoir de L'Etude Sur L'Effet de la Consommation de Lactochol ® Sur Les Parametres de L'Atherosclerose[END_REF]unpublished). The studied dose was, however, below maximum proposed use levels for this NF and safety relevant endpoints included only the level of serum enzymes aspartate transaminase (AST) and alanine transaminase levels (ALT). No effects were noted.

Although the provided human studies do not raise concern with regard to the two main constituents, bLF and bLP, the Panel notes the significant limitations of these studies (i.e. not performed with the NF, dose levels tested, limited number of endpoints) in relation to the safety of the NF.

Allergenicity

The Panel notes that the source of the NF is a recognised allergen and that, therefore, the NF is potentially allergenic.

Discussion

The NF is a mixture of basic whey proteins obtained by ion exchange chromatography from skimmed cow's milk. The main constituents are bLF, bLP and TGF b2, which share an amino acid sequence homology of approximately 70, 83 and 99%, respectively, with their analogues present in human milk. The highest estimated intake of the NF based on the proposed uses and use levels would be 24.8 mg/kg bw per day in infants and 27.8 in toddlers.

The information provided on composition, specifications, production process and stability of the NF does not raise safety concerns. Taking into account the composition of the NF and the intended use levels, the Panel considers that the consumption of the NF is not nutritionally disadvantageous.

The Panel considers that there is no concern with respect to genotoxicity of the NF. The NOAEL of a subchronic rat study was 2,000 mg/kg bw per day (the highest dose tested).

Considering the source and nature of the NF, the Panel considers the margin of exposure (MOE) of 154 to be sufficient for the adult population (on an estimated high intake of 13 mg/kg bw per day).

For infants and toddlers, the MOE would be at least 81 and 72, respectively. Noting the occurrence of bLF, bLP and TGF-b2 present in bovine-based infant formulae, the Panel considers that the increase of their intakes from the NF at the proposed use level for infant formulae would not be of concern. Taking into account the source, the production process, the nature of the NF as well as the history of consumption of the main components of the NF, and that the NOAEL in a subchronic rat study, which was the highest dose tested, the Panel considers that the MOE of 81 and 72 for infants and toddlers are sufficient.

Conclusions

The Panel concludes that the NF, whey basic protein isolate, is safe under the proposed uses and use levels.

The Panel could not have reached the conclusion on the safety of the NF under the proposed conditions of use without the data claimed as proprietary by the applicant, i.e. 90-day subchronic toxicity study by [START_REF] Silvano | 13-week toxicity study by the oral route (gavage) in rats followed by a 4-week treatment-free period[END_REF]unpublished) to determine the MOE. 3) On 22 02 2018, EFSA received a valid application from the European Commission on whey basic protein isolate as NF, which was submitted by Armor Prot eines S.A.S., and the scientific evaluation procedure started. 4) During its meeting on 27 June 2018, the NDA Panel, having evaluated the data, adopted a scientific opinion on the safety of whey basic protein isolate as a NF pursuant to Regulation (EU) 2015/2283.

Steps taken by EFSA

  Abstract................................................................................................................................................... Summary................................................................................................................................................. 1. Introduction................................................................................................................................. 1.1. Background and Terms of Reference as provided by the European Commission ................................ 2. Data and methodologies ............................................................................................................... 2.1. Data............................................................................................................................................ 2.2. Methodologies.............................................................................................................................. 3. Assessment.................................................................................................................................. 3.1. Introduction................................................................................................................................. 3.2. Identity of the NF......................................................................................................................... 3.3. Production process ....................................................................................................................... 3.4. Compositional data....................................................................................................................... 3.4.1. Stability ....................................................................................................................................... 3.5. Specifications ............................................................................................................................... 3.6. History of use of the NF and/or of its source .................................................................................. 3.6.1. History of use of the source .......................................................................................................... 3.6.2. History of use of the NF................................................................................................................ 3.7. Proposed uses and use levels and anticipated intake....................................................................... 3.7.1. Target population ......................................................................................................................... 3.7.2. Proposed uses and use levels ........................................................................................................ 3.7.3. Anticipated intake of the NF .......................................................................................................... 3.8. Absorption, distribution, metabolism and excretion (ADME) ............................................................. 3.9. Nutritional information .................................................................................................................. 3.10. Toxicological information............................................................................................................... 3.10.1. Genotoxicity................................................................................................................................. 3.10.2. Subchronic toxicity ....................................................................................................................... 3.10.3. Chronic toxicity and carcinogenicity ............................................................................................... 3.10.4. Reproductive and developmental toxicity........................................................................................ 3.10.5. Human data................................................................................................................................. 3.11. Allergenicity ................................................................................................................................. 4. Discussion ................................................................................................................................... 5. Conclusions.................................................................................................................................. Steps taken by EFSA ................................................................................................................................ References............................................................................................................................................... Abbreviations ...........................................................................................................................................

4

  EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), Turck D, Bresson J-L, Burlingame B, Dean T, Fairweather-Tait S, Heinonen M, Hirsch-Ernst KI, Mangelsdorf I, McArdle H, Naska A, Neuh€ auser-Berthold M, Nowicka G, Pentieva K, Sanz Y, Siani A, Sj€ odin A, Stern M, Tom e D, Vinceti M, Willatts P, Engel K-H, Marchelli R, P€ oting A, Poulsen M, Salminen S, Schlatter J, Arcella D, Gelbmann W, de Sesmaisons-Lecarr e A, Verhagen H and van Loveren H, 2016. Guidance on the preparation and presentation of an application for authorisation of a novel food in the context of Regulation (EU) 2015/ 2283. EFSA Journal 2016;14(11):4594, 24 pp. https://doi.org/10.2903/j.efsa.2016.4594 Safety of whey basic protein isolate www.efsa.europa.eu/

  (a):[START_REF] Pamblanco | Proteins in preterm and term milk from mothers delivering appropriate or small-for-gestational age infants[END_REF];[START_REF] Prentice | The nutritional role of breast-milk IgA and lactoferrin[END_REF];[START_REF] Hirai | Concentrations of lactoferrin and iron in human milk at different stages of lactation[END_REF];[START_REF] Hennart | Lysozyme, lactoferrin, and secretory immunoglobulin A content in breast milk: influence of duration of lactation, nutrition status, prolactin status, and parity of mother[END_REF];[START_REF] Rudloff | Protein and nonprotein nitrogen components in human milk, bovine milk, and infant formula: quantitative and qualitative aspects in infant nutrition[END_REF]. (b): Ferenc[START_REF] Levay | Lactoferrin: a general review[END_REF];[START_REF] Korhonen | Bioactive peptides: novel applications for milk proteins[END_REF]. (c):[START_REF] Srivastava | Cytokines in human milk[END_REF]. (d):[START_REF] Jouni | Concentration of transforming growth factor-beta in an infant formula from five countries and human milk from Mexico and the US[END_REF]. (e):[START_REF] Shin | Purification and quantification of lactoperoxidase in human milk with use of immunoadsorbents with antibodies against recombinant human lactoperoxidase[END_REF]. (f):[START_REF] Indyk | Determination of minor proteins of bovine milk and colostrum by optical biosensor analysis[END_REF]. (g):[START_REF] Elfstrand | Immunoglobulins, growth factors and growth hormone in bovine colostrum and the effects of processing[END_REF]. (h):[START_REF] Gauthier | Growth factors from bovine milk and colostrum: composition, extraction and biological activities[END_REF]. (i): Calculated value based on an infant formula with protein content of 14 g per 100 mL, whey to casein ratio of 60:40 as well as the content of the constituents found in cow's milk. (j):[START_REF] Jouni | Concentration of transforming growth factor-beta in an infant formula from five countries and human milk from Mexico and the US[END_REF].

1)

  Letter from the European Commission to the European Food Safety Authority with the request for a scientific opinion on the safety of ARES(2017)6052616, dated 11 December 2017. 2) As the application was re-submitted by the applicant through the e-submission portal in order for it to be treated as an application under Regulation (EU) 2015/2283 as set out in article 35(1) of Regulation (EU) 2015/2283, the mandate submitted under ARES(2017) 6052616 was replaced by the present mandate Ares(2018)994596, dated 21 February 2018.

Table 1 :

 1 Batch-to-batch analyses of the NF

	Safety of whey basic protein isolate
			Manufacturing Lot No.
	Parameter	111106	111217	130830	131114	140724
	Appearance	Yellowish	Yellowish	Yellowish	Yellowish	Yellowish
		grey powder	grey powder	grey powder	grey powder	grey powder
	pH (5% solution w/v)	6.2	6.4	6.3	6.1	6.7
	Total Protein (%)	91	93	94.3	96	94.5
	Lactoferrin (%)	32	51	50	63	70
	Lactoperoxidase (%)	32	24	25	19	17
	TGF-b2 (mg/100 g)	14.6	13.6	16.2	17.4	14
	Moisture (%)	4.6	4.1	4.8	3.7	4.2
	Lactose (%)	< 0.09	< 0.09	< 0.09	< 0.09	< 0.09
	Fat (%)	2.5	2.8	2	0.5	2
	Ash (%)	1.7	1.3	1	0.5	0.5
	Iron (mg/100 g)	18.8	19.2	17.4	16.7	16.9
	Heavy metals
	Lead (mg/kg)	< 0.02	< 0.02	< 0.02	< 0.02	< 0.02
	Cadmium (mg/kg)	< 0.005	< 0.005	< 0.005	< 0.005	< 0.005
	Mercury (mg/kg)	< 0.005	< 0.005	< 0.005	< 0.005	< 0.005
	Arsenic (mg/kg)	0.002	0.002	0.002	0.002	< 0.05
	Microbiological parameters
	Aerobic mesophilic	50	50	330	20	10
	count (CFU/g)
	Enterobacteriaceae (CFU/g)	0	0	0	0	0
	Yeasts (CFU/g)	0	0	0	0	0
	Moulds (CFU/g)	0	0	0	0	0
	Escherichia coli (in 1 g)	Negative	Negative	Negative	Negative	Negative
	Coagulase positive	Negative	Negative	Negative	Negative	Negative
	Staphylococci (in 1 g)
	Salmonella (in 25 g)	Negative	Negative	Negative	Negative	Negative
	Listeria (in 25 g)	Negative	Negative	Negative	Negative	Negative
	Cronobacter spp. (in 25 g)	Negative	Negative	Negative	Negative	Negative

www.efsa.europa.eu/efsajournal 7 EFSA Journal 2018;16(7):5360

Table 2 :

 2 Specifications of whey protein isolate

	Parameter	Limit	Method
	Appearance	Yellowish grey powder Visual Inspection
	Foreign matter	Absent	Visual inspection in 25 g (ADMI chart,
	(Scorched particles)		solubilised with 0.15 M NaCl solution)
	pH (5% solution w/v)	5.5-7.6	5% (w/v) solution, pH meter
	Total Protein (%)	≥ 90	Kjeldahl method (IDF20/ISO 8968) [N x 6.38]
	Lactoferrin (%)	25-75	HPLC (modified GB1903.17.2016)
	Lactoperoxidase (%)	10-40	HPLC (modified GB1903.17.2016)
	TGF-b2 (mg/100 g)	12-18	ELISA (Quantikine human TGF-b2, R&D
			Systems)
	Moisture (%)	≤ 6.0	ISO 5550
	Lactose (%)	≤ 3.0	Enzymatic method (Lactose/D-Galactose kit,
			Boehringer Mannheim/R-Biopharm)
	Fat (%)	≤ 4.5	AFNOR Chimie II 3B 1986
	Ash (%)	≤ 3.5	ISO 5545
	Iron (mg/100 g)	≤ 25	AAS
	Heavy metals
	Lead (mg/kg)	< 0.1	ICP-MS
	Cadmium (mg/kg)	< 0.2	ICP-MS
	Mercury (mg/kg)	< 0.6	ICP-MS
	Arsenic (mg/kg)	< 0.1	ICP-MS
	Microbiological specifications
	Aerobic mesophilic count (CFU/g)	≤ 10,000	ISO 4833
	Enterobacteriaceae (CFU/g)	≤ 10	ISO 21528-1
	Yeasts (CFU/g)	≤ 50	ISO 6611 | IDF 94:2004
	Moulds (CFU/g)	≤ 50	ISO 6611 | IDF 94:2004
	Escherichia coli (in 1 g)	Negative	ISO 7251
	Coagulase positive Staphylococci	Negative	ISO 6888-3
	(in 1 g)
	Salmonella (in 25 g)	Negative	VIDAS Easy Salmonella method (equivalent
			to ISO6579)
	Listeria (in 25 g)	Negative	VIDAS LIS method (equivalent to ISO
			11290-1/A1:2004)
	Cronobacter spp. (in 25 g)	Negative	ISO/TS 22964:2006

www.efsa.europa.eu/efsajournal 8 EFSA Journal 2018;16(7):5360

Table 3 :

 3 Contents of bLF, bLP and TGF-b2 in the human and cow's milk and infant formulae

	Item	lactoferrin (mg/100 mL)	lactoperoxidase (mg/100 mL)	TGF-b2 (lg/100 mL)

Table 4 :

 4 Proposed conditions of use of the NF

	Proposed Food use	Suggested Serving Size	Max. Use Level
	Infant formula	N/a	30 mg/100 g (a)

Table 5 :

 5 Calculated maximum use levels of bLF, bLP and TGF-b2 for the proposed food categories

			Calculated Maximum Use Level
	Proposed Food use	Suggested Serving Size	bLF	bLP	TGF-b2
			mg/100 mL	mg/100 mL	lg/100 mL
	Infant Formula (a)	n/a	2.9	1.6	0.7
	Follow-on formula (a) n/a	3.2	1.7	0.76
			mg/per serving	mg/per serving	lg/serving
	Meal replacement	250 g (100 mg of the NF)	75	40	18
	Beverages
	Foods for special	610 mg	457.5	244	110
	medical purposes	(per serving and per day)
	Food supplements	610 mg	457.5	244	110
		(per serving and per day)
	(a): Reconstituted.

Safety of whey basic protein isolate www.efsa.europa.eu/efsajournal 10 EFSA Journal 2018;16(7):5360

Table 7 :

 7 Estimated mean intakes of the NF from meal replacement beverages estimated on the basis of consumption data from the United Kingdom (UKDA, 2014)

	Safety of whey basic protein isolate
	Population group	% Users	No of Consumers	Mean consumption (mg/d) (mg/kg bw per d)
	Children	0.6	6	80.1	3.90
	4-10 years
	Female Teenagers	0.2	1	260.0	3.46
	11-18 years
	Male Teenagers	1.8	9	98.7	1.64
	11-18 years
	Female Adults	0.8	14	138.0	1.66
	19-64 years
	Male Adults	2.7	24	112.0	1.32
	19-64 years
	Adults ≥ 65 years	1.5	6	84.7	1.03

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www.efsa.europa.eu/efsajournal

Regulation (EU) No

258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and novel food ingredients. OJ L 43, 14.2.1997, p. 1-6. 2 Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods, amending Regulation (EU) No 1169/ 2011 of the European Parliament and of the Council and repealing Regulation (EC) No 258/97 of the European Parliament and of the Council and Commission Regulation (EC) No 1852/2001 (2013/0435 (COD). OJ L 327, 11.12.2015, p. 1-22. 3 Commission Implementing Regulation (EU) 2017/2469 of 20 December 2017 laying down administrative and scientific requirements for applications referred to in Article 10 of Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods. OJ L 351, 30.12.2017, pp. 64-71.

18314732, 2018, 7, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2018.5360 by Cochrane France, Wiley Online Library on [01/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

EFSA Journal 2018;16(7):5360

EFSA Journal 2018;16(7):5360 18314732, 2018, 7, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2018.5360 by Cochrane France, Wiley Online Library on [01/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License


Dominique Turck

Jean-Louis Bresson

Barbara Burlingame

Tara Dean

Susan Fairweather-Tait

Marina Heinonen

Karen Ildico

Inge Mangelsdorf

Harry J Mcardle

Androniki Naska

Monika Neuh € Auser-Berthold

Gra _ Zyna Nowicka

Kristina Pentieva

Yolanda Sanz

Alfonso Siani

Anders Sj

Martin Stern

Daniel Tom

Marco Vinceti

Peter Willatts

Karl-Heinz Engel

Rosangela Marchelli

Josef Rudolf Schlatter

Reinhard Ackerl

Henk Van Loveren

Safety of egg membrane hydrolysate as a novel food pursuant to Regulation (EU) 2015/2283 EFSA Panel on Dietetic Products, Nutrition and Allergies (EFSA NDA Panel)

Keywords: egg membrane hydrolysate, novel food, safety

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver a scientific opinion on egg membrane hydrolysate as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is a water-soluble hydrolysate of egg membrane, produced by alkaline treatment of the thin layer lining the shells of chicken eggs. The main constituents of the NF are elastin, collagen and glycosaminoglycans. The information provided on the production process, batch-to-batch variability, composition and specifications of the NF is sufficient and does not raise safety concerns. The NF is proposed to be used as a food supplement for adults, at a maximum daily amount of 450 mg. The consumption of the NF is not nutritionally disadvantageous. One human study, which was not designed for safety but included a number of endpoints pertaining to safety, did not raise safety concerns. The Panel considers that taking into account the information provided and considering the nature, the source and the production process of the NF, there are no safety concerns for the NF at the proposed conditions of use. The Panel concludes that the NF, egg membrane hydrolysate, is safe as a food supplement at a dose of 450 mg/day. The target population for the NF is the general adult population.

Summary

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver a scientific opinion on egg membrane hydrolysate as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The assessment of the safety of the NF follows the methodology set out in the EFSA Guidance on the preparation and presentation of an application for authorisation of a novel food in the context of Regulation (EU) 2015/2283 and in the Commission Implementing Regulation (EU) 2017/2469.

The NF which is the subject of the application is a water-soluble hydrolysate of egg membrane, produced by alkaline treatment of the thin layer lining the shells of chicken eggs. The NF is a protein-based, water-soluble, off-white powder. Its main constituents are elastin, collagen and glycosaminoglycans. The NF has been marketed outside the European Union (EU) under the trade name of BiovaFlex TM .

The information provided on the production process, batch-to-batch variability, composition and specifications of the NF is sufficient and does not raise safety concerns.

The NF is proposed to be used as a food supplement for adults, at a maximum daily amount of 450 mg.

Even though the alkaline treatment conditions employed during the manufacturing process will result in partial racemisation of the amino acids in the NF and, thus, their limited digestibility/ bioavailability, taking into account the proposed conditions of use the Panel considers that the consumption of the NF is not nutritionally disadvantageous.

There are no concerns for genotoxicity of the NF.

Of the two human studies provided with the NF, one study reported on safety related endpoints. This study, even though not designed for safety but including a number of endpoints pertaining to safety, did not raise safety concerns.

The Panel considers that taking into account the information provided and considering the nature, the source and the production process of the NF, there are no safety concerns for the NF at the proposed conditions of use.

The Panel concludes that the NF, egg membrane hydrolysate, is safe at the proposed conditions of use, i.e. as a food supplement at a dose of 450 mg/day. The target population is the general adult population.

Table of contents

Background and Terms of Reference as provided by the European Commission

On 5 August 2016, the company Biova LLC submitted a request to the Danish Veterinary and Food Administration (DVFA) in accordance with Article 4 of Regulation (EU) 258/1997 1 to place on the market egg membrane hydrolysate as a novel food (NF).

On 7 June 2017, the DVFA forwarded to the Commission its initial assessment report, which came to the conclusion that egg membrane hydrolysate meets the criteria for acceptance of a NF defined in Article (3)1 of Regulation (EU) 258/1997.

On 12 June 2017, the Commission forwarded the initial assessment report to the other Member States (MS). Several MS raised objections or submitted comments.

The concerns of a scientific nature raised by the MS can be summarised as follows:

• Further information was requested on the manufacturing process.

• Further details on the composition of the NF were requested.

• One MS questioned the applicant's rationale for not providing a full toxicological assessment ('based on the lack of toxicological effects of the source, i.e. egg membrane, which is occasionally consumed with boiled eggs').

• Concerns were raised by one MS concerning potential drug interactions between the NF and medication taken by people with joint pain (who were the targeted study subjects in the submitted human study and who might potentially be the target population for the NF).

2.

Data and methodologies

Data

The safety assessment of this NF is based on data supplied in the application, the initial assessment by the DVFA, the concerns and objections of a scientific nature raised by the other MS and the responses from the applicant thereto.

The information in support of this application has been submitted through the Commission e-submission portal (NF 2018/0124).

Administrative and scientific requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in the Commission Implementing Regulation (EU) 2017/2469 3 .

A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of a NF application. 4 As indicated in this guidance, it is the duty of the applicant to provide all of the available (proprietary, confidential and published) scientific data, including both data in favour and not in favour to supporting the safety of the proposed NF.

This NF application includes a request for the protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283. The data requested by the applicant to be protected comprise: detailed description of the manufacturing process, GRAS dossier [START_REF] Biova | The Generally Recognized as Safe (GRAS) Status of BiovaFlex[END_REF], proteomic analysis, bacterial reverse mutation test (ST&T Consultants, 2009b, unpublished), in vitro micronucleus assay [START_REF] Roy | In vitro mammalian cell micronucleus assay in TK6 cells[END_REF]unpublished), acute oral toxicity study (ST&T Consultants, 2009a, unpublished), human study (ST&T Consultants, 2009c, unpublished), RAST-inhibition assay (Food Allergy Research and Resource Program, 2008a, unpublished), quantitative egg allergen test (Food Allergy Research and Resource Program, 2008b, unpublished) and a guinea pig sensitisation assay (ST&T Consultants, 2009d, unpublished).

Methodologies

The assessment follows the methodology set out in the EFSA guidance on NF applications and the principles described in the relevant existing guidance documents from the EFSA Scientific Committee. The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 and in Article 7 of the Commission Implementing Regulation (EU) 2017/2469. This assessment concerns only risks that might be associated with the consumption of the NF under the proposed conditions of use, and it is not an assessment of the efficacy of the NF with regard to any claimed benefit.

Assessment

Introduction

The NF which is the subject of the application is a water-soluble hydrolysate of egg membrane, produced by alkaline treatment of the thin layer lining the shells of chicken eggs. The powder is proposed to be used as a food supplement. The NF has been marketed outside the EU under the trade name of BiovaFlex TM .

Identity of the NF

The NF, i.e. egg membrane hydrolysate, is a protein-based, water-soluble, off-white powder. Its main constituents are elastin, collagen and glycosaminoglycans.

The NF is derived from the eggs of farmed chicken hens (Gallus domesticus).

Production process

The NF is produced according to Good Manufacturing Practice (GMP) and Hazard Analysis Critical Control Points (HACCP) principles.

The starting material for the production of the NF is eggshells from farmed chicken eggs produced for human consumption. Information on the supplier of the eggshells has been provided. It is a United States Department of Agriculture (USDA)-inspected egg-breaking facility located in the US.

The eggshells undergo hydromechanical separation in order to obtain the egg membranes, which are then further processed using a patented solubilisation method. For the processing, pH and temperature are increased and the egg membranes are kept under these conditions for a specified amount of time. No solvents other than water are used. Details on the manufacturing process including flow charts have been provided. Following the solubilisation process, the solution is filtered, concentrated, spray-dried and packaged.

The Panel notes that the conditions employed during the manufacturing process (heat treatment under alkaline pH) are expected to result in the formation of lysinoalanine as well as partial racemisation of amino acids in the NF. However, taking into account the proposed daily intake of the NF (i.e. 450 mg) and considering the dietary background intake from other processed protein-based foods, the potential intake of lysinoalanine or D-amino acids via the NF is not considered to be of safety concern.

The Panel considers that the production process is sufficiently described and does not raise safety concerns.

3.4.

Compositional data

The NF consists primarily of proteins, peptides and amino acids (about 90%), with the remainder being moisture (≤ 9%), ash (≤ 8%), fat (~1.8%), fibre (2%) and traces of carbohydrates (< 0.01%).

The most abundant proteins in the NF are elastin (~25%) and collagen (~22%). Also the fraction of glycosaminoglycans constitutes a sizable part (~18%) of the NF (values are the means of the batches provided).

In order to confirm that the manufacturing process is reproducible and adequate to produce on a commercial scale a product with certain characteristics, the applicant provided analytical information for three batches of the NF (see Table 1).

Information was provided on the accreditation (i.e. ISO/IEC 17025:2005general requirements for the competence of testing and calibration laboratories) of the laboratory that conducted the analyses.

Additionally, the applicant provided the analytical results for a number of pesticides, dioxins and antibiotics in the NF, which were all below the limit of detection.

Following a request from a MS for more details on the characterisation of the NF, the applicant submitted information on the total amino acid composition of the NF and, in addition, compared it to that of native egg membrane. Differences in the content of a number of amino acids were observed, which might be owing to the hydrolysation step that occurs during the manufacturing process of the NF. For example, the amide groups of glutamine and asparagine residues are partially hydrolysed during the manufacturing process, increasing the content of glutamic acid and aspartic acid in the NF compared to native egg membrane. Furthermore, the drying step might dehydrate part of the hydroxyproline in the native egg membrane to proline, leading to an increase of proline and a decrease of hydroxyproline in the NF. The observed decrease of cystine in the NF might be owing to a loss during processing prior to amino acid analysis.

The Panel considers that the information provided on the composition of the NF is sufficient and does not raise safety concerns.

Stability

In order to demonstrate the stability of the NF over the intended shelf life, specified as a period of 48 months in a cool and dry environment, the applicant provided the results of real-time stability testing for one batch over 48 months at warehouse room temperature (relative humidity not provided). The results for the parameters analysed (i.e. nitrogen-containing compounds, collagen, elastin and total glycosaminoglycans; moisture, water activity, microbial growth) remained within the limits set in the specifications.

Following a request from a MS for additional information concerning the stability of the NF, the applicant provided the results of two additional batches, even though only up to 19.9 and 25.5 months, respectively. Over this time frame, the analysed parameters remained within the limits set in the specifications.

The Panel considers that the data provided sufficient information with respect to the stability of the NF up to 20 months.

Specifications

The specifications of the NF are indicated in Table 2. They comprise the content of collagen, elastin, and total glycosaminoglycans as well as other physicochemical parameters, limits for contaminants such as heavy metals and microbiological specifications. The Panel considers that the information provided on the specifications of the NF is sufficient and does not raise safety concerns.

3.6.

History of use of the NF and/or of its source

History of use of the source

The NF is produced from chicken eggs (Gallus domesticus), which have a long history of food use within the EU.

Even though not consumed as a food as such, egg membranes may incidentally be consumed with boiled eggs.

History of use of the NF

In 2009, the NF was notified as a New Dietary Ingredient (NDI) in the United States. Since then approximately 17,000 kg of the NF have been sold (under the tradename of BiovaFlex ® ) in the US.

In 2015, the NF (i.e. BiovaFlex ® ) was notified by the applicant to be Generally Recognized as Safe (GRAS) for use in food and food supplements [START_REF] Biova | The Generally Recognized as Safe (GRAS) Status of BiovaFlex[END_REF].

According to the information provided, no adverse reactions to egg membrane products on the US market have been reported to date under the US FDA's MedWatch adverse event reporting programme.

3.7.

Proposed uses and use levels

Target population

The target population proposed by the applicant is adults above 35 years of age.

Proposed uses and use levels

The applicant proposed to use the NF as an ingredient in food supplements, at a maximum dose of 450 mg per day.

Absorption, distribution, metabolism and excretion (ADME)

No ADME data have been provided on the NF.

Nutritional information

The applicant provided a nutritional analysis of the NF, which comprises information on the content of nitrogen-containing compounds, fibre, sodium, carbohydrates, fatty acids, vitamins and minerals in the NF.

The NF is primarily (about 90%) composed of nitrogen-containing compounds (i.e. proteins, peptides and amino acids). The proposed daily amount of 450 mg of the NF would result in an additional intake of approximately 400 mg nitrogen-containing compounds per day.

The Panel notes that the alkaline treatment conditions employed during the manufacturing process will result in partial racemisation of the amino acids in the NF and, consequentially, in limited digestibility/bioavailability of such amino acids.

However, taking into account the proposed conditions of use (i.e. 450 mg/day), the Panel considers that the consumption of the NF is not nutritionally disadvantageous. 3.10.

Toxicological information 3.10.1. Genotoxicity

The applicant provided a bacterial reverse mutation assay (ST&T Consultants, 2009b, unpublished) and an in vitro micronucleus assay (Roy, 2016, unpublished).

The bacterial reverse mutation assay (ST&T Consultants, 2009b, unpublished) was described as following a non-GLP protocol. Reference was made to the publications by [START_REF] Ames | Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test[END_REF] and [START_REF] Maron | Revised methods for the Salmonella mutagenicity test[END_REF]. For the assay five different strains of Salmonella Typhimurium were used, i.e. TA97a, TA98, TA100, TA102 and TA1535. The NF, which was found to be completely soluble in sterile water at 50 mg NF/mL (used as a stock solution), was tested at concentrations up to 5,000 lg/plate. The testing included negative (i.e. solvent) and positive controls and was carried out with and without metabolic activation (i.e. S9-mix from rat liver derived from Aroclor TM -treated rats). All tests were carried out in triplicate. The spontaneous reversion rates remained within accepted values for each strain. All the five strains were sensitive to the mutagens used for the positive controls. No cytotoxicity was observed. There was no increase in the number of revertant colonies following exposure to the NF at concentrations up to 5,000 lg/plate in the presence or absence of metabolic activation.

The in vitro micronucleus assay (Roy, 2016, unpublished), which was conducted in compliance with US FDA GLP Regulations, was carried out in a lymphocytic cell line (TK6) of human origin. The batch of the NF used in the study was within the specifications set in section 3.5. As vehicle water was used. In a preliminary test, the TK6 cells were exposed to vehicle alone and to nine different concentrations of the NF with half-log dose spacing. Based on the findings of the pretest, the doses of the NF chosen for the definitive assay were 100, 240, 350 and 500 lg/mL for each treatment condition. For the assay, the cells were exposed to the NF for 4 h with and without S9-activation system, and for 27 h without the S9-mix. No cytotoxicity (defined as ≥ 50% decrease in population doubling relative to vehicle control) was observed at any dose level. The dose levels selected for microscopic analysis were 240, 350 and 500 lg/mL for each treatment condition. A minimum of 1,000 mononucleate cells from each culture were examined and scored for the presence of micronuclei. The frequency of micronuclei in cells exposed to the NF was not significantly increased at any dose level with or without metabolic activation.

The Panel considers that taking into account the test results provided and considering the nature, the source and the production process of the NF there are no concerns regarding genotoxicity of the NF.

Toxicity studies

The applicant submitted one acute oral toxicity study (ST&T Consultants, 2009a, unpublished), which was carried out in Sprague-Dawley albino rats. The rats (5/sex per group) were administered a single oral dose of the NF at a dose of 5,000 mg/kg body weight (bw). The animals were observed at 1, 3, 6 and 24 h after treatment, and daily thereafter for a total of 14 days. All animals survived the 14-day observation period. The oral median lethal dose was thus concluded to be greater than 5,000 mg/kg bw.

No other toxicity studies were provided.

Human data

Two human studies with the NF were provided (ST&T Consultants, 2009c, unpublished;[START_REF] Jensen | Support of joint function, range of motion and physical activity levels by consumption of a water-soluble egg membrane hydrolysate[END_REF] one of which reported on safety related endpoints (ST&T Consultants, 2009c, unpublished).

This trial (ST&T Consultants, 2009c, unpublished) was an open-label uncontrolled pilot study conducted in 42 subjects of at least 18 years with a history of arthritic-type joint pain or connective tissue disorder with a reduced range of knee motion. The study subjects were instructed to consume for 6 weeks 450 mg/day of the NF. The primary endpoint of the study was to assess a potential effect of the NF on pain, stiffness, and discomfort of knee-associated musculoskeletal disorders. As secondary endpoint the safety of the NF was investigated. Health evaluations of the study subjects were performed at baseline and at weeks 2, 4 and 6. Adverse events and compliance were recorded by study subjects in diaries and were reviewed at each visit. As part of the safety protocol, blood chemistry, haematology and allergen antibody assays (i.e. concentration of immunoglobulin E (IgE) antibodies to egg allergens) were performed. The haematology analysis included complete blood cell counts and concentrations of haematocrit and haemoglobin as well as cell morphology. The blood chemistry analysis comprised non-fasting glucose, sodium, potassium, chloride, carbon dioxide, urea nitrogen, creatinine, calcium, alkaline phosphatase, total protein, albumin and globulin. In addition, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin concentrations were analysed to monitor liver function. Twelve subjects dropped out from the study. The reasons were provided (i.e. personal (5), loss-to-follow up (2), high blood pressure (2), egg allergy (2) and 'pressure' (1)). The blood parameters analysed were all within normal ranges. No statistically significant changes from baseline were observed. No significant increases in the concentration of IgE antibodies to egg allergens were noticed in any subject. No adverse events were reported.

Allergenicity

In order to determine the allergenic potential of the NF, a radioallergosorbent test (RAST)-inhibition assay was performed (Food Allergy Research and Resource Program, 2008a, unpublished), which is a competitive radioimmunoassay that uses serum from egg-allergic individuals and which detects IgEbinding (as described by [START_REF] Hefle | Adverse reaction to lupine-fortified pasta[END_REF]. The RAST-inhibition curve of the NF was rather flat when compared to the curve of a whole egg sample (slope of 1.65 vs 23.42 for whole egg), indicating little inhibition (i.e. binding) of egg specific-IgE by the NF.

The allergenicity of the NF was further investigated using the Neogen Veratox ® Quantitative Egg Allergen Test, which is specific for egg white. In three batches of the NF analysed, the amount of egg white was below the limit of detection (i.e. 2.5 mg/kg). In a control sample of concentrated egg membrane not having undergone solubilisation, the amount of egg white was 2,370 mg/kg (Food Allergy Research and Resource Program, 2008b, unpublished).

The applicant also submitted a guinea pig sensitisation assay (Buehler), in which the topical application of the NF to the skin of 12 Hartley albino guinea pigs for three weeks did not result in any evidence of skin irritation or sensitisation (ST&T Consultants, 2009d, unpublished).

The applicant informed that notwithstanding the above results the NF will be labelled in accordance with Regulation No 1169/20115 , which requires labelling for substances or products with a potential to cause allergies or intolerances (such as eggs and products thereof).

The Panel notes that the source of the NF is a recognised allergen and that, therefore, the NF is potentially allergenic.

Discussion

The NF which is the subject of the application is a hydrolysate of egg membrane.

The information provided on the composition, the specifications, the production process and the batch-to-batch variability of the NF is sufficient and does not raise safety concerns.

The intention is to market the NF as a food supplement to adults, with a proposed maximum intake of 450 mg/day.

The human study, which was not designed for safety but included a number of endpoints pertaining to safety, did not raise safety concerns.

There are no concerns for genotoxicity of the NF. The Panel considers that taking into account the information provided and considering the nature, the source and the production process of the NF, there are no safety concerns for the NF at the proposed conditions of use.

Conclusions

The Panel concludes that the NF, egg membrane hydrolysate, is safe at the proposed conditions of use, i.e. as a food supplement at a dose of 450 mg/day. The target population is the general adult population.

The Panel could not have reached its conclusion on the safety of the NF under the proposed conditions of use without the detailed description of the manufacturing process for which data protection was requested by the applicant.

  Abstract..................................................................................................................................................... Summary................................................................................................................................................... 1. Introduction.................................................................................................................................... 1.1. Background and Terms of Reference as provided by the European Commission ................................... 2. Data and methodologies .................................................................................................................. 2.1. Data............................................................................................................................................... 2.2. Methodologies................................................................................................................................. 3. Assessment..................................................................................................................................... 3.1. Introduction.................................................................................................................................... 3.2. Identity of the NF............................................................................................................................ 3.3. Production process .......................................................................................................................... 3.4. Compositional data.......................................................................................................................... 3.4.1. Stability .......................................................................................................................................... 3.5. Specifications .................................................................................................................................. 3.6. History of use of the NF and/or of its source ..................................................................................... 3.6.1. History of use of the source ............................................................................................................. 3.6.2. History of use of the NF................................................................................................................... 3.7. Proposed uses and use levels ........................................................................................................... 3.7.1. Target population ............................................................................................................................ 3.7.2. Proposed uses and use levels ........................................................................................................... 3.8. Absorption, distribution, metabolism and excretion (ADME) ................................................................ 3.9. Nutritional information ..................................................................................................................... 3.10. Toxicological information.................................................................................................................. 3.10.1. Genotoxicity.................................................................................................................................... 3.10.2. Toxicity studies................................................................................................................................ 3.10.3. Human data.................................................................................................................................... 3.11. Allergenicity .................................................................................................................................... 4. Discussion ...................................................................................................................................... 5. Conclusions..................................................................................................................................... Steps taken by EFSA .................................................................................................................................. References................................................................................................................................................. Abbreviations .............................................................................................................................................

  Pursuant to Article 35(1) of Regulation (EU) 2015/2283 2 , any request for placing a novel food on the market within the Union submitted to a Member State in accordance with Article 4 of Regulation (EC) No 258/97 of the European Parliament and of the Council concerning novel foods and novel foods ingredients and for which the final decision has not been taken before 1 January 2018, shall be treated as an application submitted under Regulation (EU) 2015/2283. On 20 April 2018, the European Commission asked EFSA to provide a scientific opinion by carrying out the additional assessment for egg membrane as a novel food ingredient, in accordance with Article 10(3) of Regulation (EU) 2015/2283.

  Association for Clinical Chemistry; CFU: colony forming units; CMMEF: Compendium of Methods for the Microbiological Examination of Foods; ELISA: enzyme-linked immunosorbent assay; FDA BAM: United States Food and Drug Administration's Bacteriological Analytical Manual; ICP-MS: inductively coupled mass spectrometry; MPN: most probable number; USP: United States Pharmacopeia.

Table 1 :

 1 Batch-to-batch analysis of the NF

		Batch results of three commercial lots
	Parameter	P3095	P4132	P4252-1
	Chemical
	Total nitrogen-containing compounds (%)	90.79	89.69	89.47
	Collagen (%)	20	22	25
	Elastin (%)	26	26	22
	Total glycosaminoglycans (%)	12.7	19.8	21
	Calcium (%)	0.14	0.36	0.42
	Physical
	pH	6.6	6.9	6.8
	Ash (%)	5.28	6.68	5.69
	Moisture (%)	6.6	6.95	7.4
	Water activity	0.207	0.246	0.244
	Solubility (in water)	Soluble	Soluble	Soluble
	Bulk density (g/cc)	0.66	0.66	0.63
	Heavy metal(s)
	Arsenic (mg/kg)	0.02	0.02	0.02
	Lead (mg/kg)	0.02	0.03	0.09
	Cadmium (mg/kg)	0.01	0.01	0.01
	Mercury (mg/kg)	0.01	0.01	0.01
	Microbiology
	Aerobic plate count (CFU/g)	150	60	90
	Escherichia coli (MPN/g)	< 3	< 3	< 3
	Salmonella (in 25 g)	negative	negative	negative
	Coliforms (MPN/g)	< 3	< 3	< 3
	Staphylococcus aureus (CFU/g)	< 10	< 10	< 10
	Mesophilic spore count (CFU/g)	20	20	20
	Thermophilic spore count (CFU/10 g)	< 10	< 10	< 10
	Yeast (CFU/g)	< 10	< 10	< 10
	Mould (CFU/g)	< 10	< 10	10
	CFU: colony forming units; MPN: most probable number.

Safety of egg membrane hydrolysate as a NF www.efsa.europa.eu/efsajournal 7 EFSA Journal 2018;16(7):5363

Table 2 :

 2 Specifications of the NF

	Parameter	Limits	Method
	Chemical
	Total nitrogen-containing compounds	≥ 88%	Combustion according to AOAC 990.03 and AOAC
			992.15
	Collagen	≥ 15%	Sircol TM Soluble Collagen Assay (ELISA)
	Elastin	≥ 20%	Fastin TM Elastin Assay (ELISA)
	Total glycosaminoglycans	≥ 5%	USP26 (chondroitin sulphate K0032 method)
	Calcium	≤ 1%	AOAC 965.17/985.01 mod.
	Physical
	pH	6.5-7.6	10% solution of NF at 25°C
	Ash	≤ 8%	AOAC 942.05
	Moisture	≤ 9%	AOAC 934.01
	Water activity	≤ 0.3	Vapour pressure at 25°C
	Solubility (in water)	soluble	10% solution at 25°C
	Bulk density	≥ 0.6 g/cc	USP 616
	Heavy metals
	Arsenic	≤ 0.5 mg/kg	ICP-MS according to AOAC 986.15
	Microbiological
	Aerobic plate count	≤ 2,500 CFU/g	CMMEF, 4th Ed., 2001, Method 7.6
	Escherichia coli	≤ 5 MPN/g	FDA BAM 8th Ed., ch4
	Salmonella	Negative (in 25 g) AOAC 2003.09
	Coliforms	≤ 10 MPN/g	FDA BAM 8th Ed., ch4
	Staphylococcus aureus	≤ 10 CFU/g	FDA BAM 8th Ed., ch12
	Mesophilic spore count	≤ 25 CFU/g	AACC 4240
	Thermophilic spore count	≤ 10 CFU/10 g	AACC 4240

www.efsa.europa.eu/efsajournal 8 EFSA Journal 2018;16(7):5363

www.efsa.europa.eu/efsajournal

EFSA Journal 2018;16(7):5363 18314732, 2018, 7, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2018.5363 by Cochrane France, Wiley Online Library on [01/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

EFSA Journal 2018;16(7):5363

Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/ EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No

608/2004. Official Journal of the European Union 2011, 54: 18-63.

Steps taken by EFSA


Carlo G Camarda
email: carlo-giovanni.camarda@ined.fr

María Durbán

Coherent cause-specific mortality forecasting via constrained penalized regression models

Keywords: Cause of death, Constraints, Forecasting, Mortality, Penalized Likelihood

HAL is

Introduction

Overall mortality trends are the summation of cause-specific mortality experiences. Consequently modelling and forecasting changes in cause of death patterns allows us to recognize the drivers of all-cause mortality and identify emerging health challenges. On the one hand, early literature has argued that all-cause mortality projections based on cause-specific mortality present serious drawbacks [START_REF] Wilmoth | Are mortality projections always more pessimistic when disaggregated by cause of death?[END_REF]. On the other, some approaches for forecasting cause-specific mortality has been recently proposed, though either based on the Lee-Carter model and for specific cause [START_REF] Kjaergaard | Forecasting causes of death by using compositional data analysis: the case of cancer deaths[END_REF] or on a Bayesian hierarchical model aiming to forecast cause-specific death rates for geographic subunits [START_REF] Foreman | Small Area Forecasts of Cause-Specific Mortality: Application of a Bayesian Hierarchical Model to US Vital Registration Data[END_REF].

When dealing with cause-specific mortality, we need to ensure that cause-specific deaths must sum to the total number of deaths. In the following, we model logmortality in a Poisson setting for each cause assuming only smoothness over age and time. The summation constraint thus become non linear with respect to the estimated coefficients. We embed the whole approach in a Generalized Linear Array Model framework (GLAM, [START_REF] Currie | Generalized linear array models with applications to multidimensional smoothing[END_REF] in which Lagrangian multipliers are iteratively updated to enforce constraints.

The model

We have deaths, and exposures to the risk of death, arranged in two three-dimensional arrays, Y = (y ijk ) and E = (e ijk ), each m × n × k, whose rows, columns and layers are classified by age at death (a), year of death (t) and cause of death (c). For ensuring coherence in the model, the final layer of Y contains total number of deaths (that is, k =number of causes of death+1). Note that each layer in E includes the same age-year matrix: we are in a competing risk setting. We assume that the number of deaths y ijk is Poisson distributed with mean µ ijk e ijk . The value of µ ijk , commonly named force of mortality, is the object of all mortality models.

In the following we will illustrate the method for United States, males by age-groups 30-34, . . ., 95-99, 100+, years 1978-2018 and the following five coherent groups of causes of deaths: Cardio-vascular diseases, Neoplasms, External causes, Diseases of the respiratory system and Other diseases (Human Cause-of-Death Database, 2023). We forecast total and cause-specific mortality up to 2040.

With k -1 causes of death, we deal with a three-dimensional setting. The vectorized linear predictor is given by η = Bα where the design matrix is

B = I k ⊗ B t ⊗ B a .
We use a rich basis of B-splines for age and year, and smooth surfaces are then obtained by marginal penalization. With no summation constraint the model simply reduces to a series of two-dimensional GLAMs.

To hinder singularity issues in the resulting scoring algorithm, we enforce our constraint for a large number of equally-spaced data-points. Smoothness will guarantee the remaining coherence between cause-specific and overall mortality. The penalized 3D GLAM is then subject to

C exp(η) = 0 (1)
where C sums up, for the large selected number of age and years, cause-specific deaths and then subtract the associated total number of deaths. The matrix C can be written as a Kronecker product C = C c ⊗ C t ⊗ C a and therefore, as for the linear functions and the inner products within the scoring algorithm, it can be included as a sequence of nested matrix operations in a GLAM framework.

The use of Lagrange multipliers ω for each age-year ensures that the constraint is enforced, yielding the following constrained penalized Poisson log-likelihood:

� P = y � Bα -e � exp(Bα) - 1 2 α � P α -ω � CE exp(Bα). (2)
We compute the derivatives of (2), and by means of Newton-Raphson we find the following scoring algorithm:

� B T W B + P + B T diag(C T ω) Ṽ B B T Ṽ C T C Ṽ B 0 � � α ω � = � B T W z + B T diag(C T ω) Ṽ B α C Ṽ B α -Cγ � (3)
where γ = exp η, V = diag(γ). W and z are the Poisson regression weights and working response, respectively. The penalty P ensures smoothness over age and time for each cause and it has a block-diagonal structure.

In order to handle k -1 causes of death across different age groups and years, we face the challenge of optimizing 2 • k smoothing parameters. To avoid dealing with such a high-dimensional optimization problem, we decided to utilize the smoothing parameters optimized by BIC when estimating each cause-specific age-time matrix independently.

Confidence intervals for the estimated mortality surface are calculated by stepping into the Bayesian framework, therefore:

Var(B α) = BRB T ,
where R is the top left block of the inverse of the matrix on the left hand side of (3). Of course, if there are no constraints, i.e ω = 0, we obtain the usual expression of the variance.

As in [START_REF] Currie | Smoothing and Forecasting Mortality Rates[END_REF] we treat forecasting as a missing value problem and we add shape constraints to enforce future cause-specific mortality patterns to lie within a range of valid profiles computed from observed trends [START_REF] Camarda | Smooth constrained mortality forecasting[END_REF].

Results

We fitted the proposed model to the US male mortality data described in the previous section. The left panel of Figure 1 shows actual, estimated and forecast log-mortality for a selected age (50) over years along with their 95% confidence intervals. The proposed model is able to well described historical cause-specific patterns as well as to reasonably extrapolate them into the future. The right panel of Figure 1 presents estimated death counts for a specific year (2000). Here one can easily acknowledge the equality between the sum of cause-specific deaths and the total number of deaths which is enforced by the non linear set of constraints in (1). Equally satisfactory outcomes are achieved for all ages, years and causes of deaths. In Figure 2, a commonly used summary indicator is presented: life expectancy, here at the starting age of 30. We compare our model with a estimates obtained on overall mortality using P -splines with shape constraints [START_REF] Camarda | Smooth constrained mortality forecasting[END_REF]. While the fitted values for the observed time-window are practically identical, noticeable differences emerge when making forecasts. When accounting for cause-specific patterns and ensuring that cause-specific deaths add up to the total number of deaths, the modeling of mortality yields slightly more pessimistic prospects: remaining life expectancy in 2040 at age 30 is forecast to 50.67 years in our model compared to 51.16 years in the alternative approach.

Of even greater significance is the notable reduction in the confidence intervals, indicating a significant decrease in future uncertainty around overall mortality when cause-specific trends are incorporated into the model. This outcome was expected, given the inclusion of substantial information through the incorporation of what we referred to as the summation constraint.

Conclusions

In the presented study, we propose a novel approach to model and forecast causespecific mortality. By combining penalized likelihood and iteratively computed Lagrangian multipliers we obtain smooth cause-specific mortality surfaces over ages and time, and we simultaneously enforce the necessary constraints in this setting: cause-specific deaths sum to the total number of deaths. Forecasting comes naturally in this setting and constraints are satisfied into future years, too. Additional shape are necessary to demographically informed projected patterns. Observed, estimated and forecast life expectancy at age 30 along with associated 95% confidence intervals. Proposed model estimating cause-specific and overall patterns with summation and shape constraints is compared with a simpler approach without summation constraint. USA, males, ages 30-100, years 1978-2018, forecast up to 2040.

In the example analysis of US mortality, focusing on the five leading causes of death, the proposed model yields lower overall future life expectancy compared to similar models. Additionally, it exhibits remarkably narrower future uncertainty.

Moving forward, we intend to investigate other scenarios where coherence constraints may be required, such as mortality by region, sex, and other factors. Furthermore, we have plans for a comprehensive validation study to evaluate the accuracy of future point estimates and the coverage of associated confidence intervals.

Last, Covid-19 pandemic clearly taught us that forecast future mortality by extrapolating past trends can no longer be tacitly assumed. Accounting for the impact of such short-term shocks will be a clear challenge for further mortality forecasting research as well as for our model.

FIGURE 1 :

 1 FIGURE 1: Left panel: observed, estimated and forecast cause-specific mortality rates over years for age 50 (logscale) along with associated 95% confidence intervals. Right panel: Estimated cause-specific death counts over age-groups for year 2000. In both panels, total mortality and total number of deaths are plotted. USA, males, ages30-100, years 1978-2018, forecast up to 2040.