Nutritional safety and suitability of a specific protein hydrolysate derived from sources of skimmed cow's milk andwhey protein concentrates and used in infant and follow-onformula manufactured from hydrolysed protein by Healthcare Reckitt B.V.

EFSA Journal. 2025;23:e9278. | 1 of 11 https://doi.org/10.2903/j.efsa.2025.9278 efsa.onlinelibrary.wiley.com/journal/1831-4732 SCIENTIFIC OPINION Nutritional safety and suitability of a specific protein hydrolysate derived from sources of skimmed cow's milk and whey protein concentrates and used in infant and followon formula manufactured from hydrolysed protein by Healthcare Reckitt B.V. EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) | Torsten Bohn | Montaña Cámara | Jacqueline Castenmiller | Stefaan de Henauw | KarenIldico HirschErnst | Angeles Jos | Alexandre Maciuk | Inge Mangelsdorf | Breige McNulty | Androniki Naska | Kristina Pentieva | Alfonso Siani | Frank Thies | Laurence Castle | Mary Fewtrell | Hildegard Przyrembel | Ariane Titz | Dominique Turck Adopted: 29 January 2025 DOI: 10.2903/j.efsa.2025.9278 Abstract The European Commission (EC) requested the European Food Safety Authority to evaluate the nutritional safety and suitability of a specific protein hydrolysate derived from sources of skimmed cow's milk (including nonfat) and whey protein concentrates for use in infant and followon formula manufactured by Healthcare Reckitt B.V. Healthcare Reckitt B.V. submitted a dossier to the EC seeking an amendment to Regulation (EU) 2016/127 regarding the protein sources permissible in the manufacture of infant and/or followon formula. The protein hydrolysate under evaluation is sufficiently characterised with respect to the hydrolysed protein. In the pertinent intervention study provided, an infant formula manufactured from the protein hydrolysate with a protein content of 2.3 g/100 kcal and consumed as the sole source of nutrition for 3 months led to growth that is similar to an infant formula manufactured from intact cow's milk protein with a protein content of 1.9 g/100 kcal. Data on adverse events and tolerance of the formula did not raise any concerns. No experimental data have been provided on the nutritional safety and suitability of this protein source in followon formula. Given that followon formula is consumed with complementary foods and the protein source is nutritionally safe and suitable in an infant formula that can be the sole source of nutrition of young infants, the Panel considers that the protein hydrolysate is also a nutritionally safe and suitable protein source for use in followon formula. The Panel concludes that the protein hydrolysate under evaluation is a nutritionally safe and suitable protein source for use in infant and followon formula, as long as the formula in which it is used contains a minimum of 2.3 g/100 kcal protein and complies with the compositional criteria of Regulation (EU) 2016/127 and the amino acid pattern in its Annex IIIA. KEYWORDS characterisation, clinical trial, followon formula, infant formula, nutritional safety, protein hydrolysate, suitability This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made. © 2025 European Food Safety Authority. EFSA Journal published by Wiley-VCH GmbH on behalf of European Food Safety Authority. Correspondence: [email protected] The declarations of interest of all scientific experts active in EFSA's work are available at https:// open. efsa. europa. eu/ experts 2 of 11 | NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE CONTENTS Abstract................................................................................................................................................................................................................................1 1. Introduction ..............................................................................................................................................................................................................3 1.1. Background and Terms of Reference as provided by the requestor .........................................................................................3 1.1.1. Background ....................................................................................................................................................................................3 1.1.2. Terms of Reference.......................................................................................................................................................................3 1.2. Interpretation of the Terms of Reference ............................................................................................................................................4 2. Data and Methodologies ......................................................................................................................................................................................4 2.1. Data ...................................................................................................................................................................................................................4 2.2. Methodologies..............................................................................................................................................................................................5 3. Assessment ................................................................................................................................................................................................................5 3.1. Characterisation of the protein hydrolysate ......................................................................................................................................5 3.2. Characterisation of the formula manufactured from the protein hydrolysate used in the clinical studies provided......................................................................................................................................................................................................................7 3.3. Safety and suitability of the infant and followon formula ..........................................................................................................7 3.4. Uncertainties related to the nutritional safety and suitability of the infant formula ..........................................................9 4. Conclusions ................................................................................................................................................................................................................9 5. Documentation as provided to EFSA ...............................................................................................................................................................9 6. Steps taken by EFSA ................................................................................................................................................................................................9 Abbreviations ....................................................................................................................................................................................................................9 Acknowledgements ..................................................................................................................................................................................................... 10 Requestor ......................................................................................................................................................................................................................... 10 Question number ..........................................................................................................................................................................................................10 Copyright for nonEFSA content.............................................................................................................................................................................. 10 Panel members ..............................................................................................................................................................................................................10 Legal notice .....................................................................................................................................................................................................................10 References.........................................................................................................................................................................................................................11 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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 | 3 of 11 NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE 1 | INTRODUCTION 1.1 | Background and Terms of Reference as provided by the requestor 1.1.1 | Background Commission Directive 2006/141/EC1 lays down harmonised rules applicable in the entire EU to infant formulae and followon formulae. The Directive allows the use of protein hydrolysates as source of protein in infant formulae and followon formulae under certain conditions (Articles 5–7; Annex I, point 2.2; Annex II, point 2.2 and Annex VI). Commission Delegated Regulation (EU) 2016/1272 transfers the existing rules of Directive 2006/141/EC under the new framework of Regulation (EU) No 609/2013 of the European Parliament and of the Council3 and revises them, based on the opinion of the European Food Safety Authority (EFSA) of 2014.4 In that opinion, EFSA noted that ‘the safety and suitability of each specific formula containing protein hydrolysates has to be established by clinical studies. Information on protein sources and the technological processes applied should also be provided. In this context, the Panel notes that one particular formula containing partially hydrolysed whey protein has been evaluated for its safety and suitability by the Panel (…) and has been authorised for use by Directive 2006/141/EC’. EFSA also noted that ‘the criteria given in Directive 2006/141/EC alone are not sufficient to predict the potential of a formula to reduce the risk of developing allergy to milk proteins. Clinical studies are necessary to demonstrate if and to what extent a particular formula reduces the risk of developing shortand longterm clinical manifestations of allergy in atrisk infants who are not exclusively breast fed’. Taking into account EFSA's opinion, the Delegated Regulation establishes that infant formula and followon formula manufactured from protein hydrolysates should only be allowed to be placed on the market if their composition corresponds to the one positively assessed by EFSA so far and prohibits the use of health claims describing the role of infant formula in reducing the risk of developing allergy to milk proteins. The requirements of Commission Delegated Regulation (EU) 2016/127 shall apply to infant formula and followon formula manufactured from protein hydrolysates from 22 February 2021. Pursuant to Recital 21 of the Regulation, these requirements may be amended in the future in order to allow the placing on the market of formulae manufactured from protein hydrolysates with a composition different from the one already positively assessed, following a casebycase evaluation of their safety and suitability by EFSA. In addition, if, after the assessment by EFSA, it is demonstrated that a specific formula manufactured from protein hydrolysates reduces the risk of developing allergy to milk proteins, further consideration will be given to how to adequately inform parents and caregivers about that property of the product. The requirements of Commission Delegated Regulation (EU) 2016/127 shall apply to infant formula and followon formula manufactured from protein hydrolysates from 22 February 2021. It can be expected that, before that date, dossiers on formulae manufactured from protein hydrolysates will be presented by food business operators for assessment by EFSA with a view to request possible modifications to the conditions applicable to these products in the Delegated Regulation. In this context, it is considered necessary to ask EFSA to provide scientific advice to the Commission on dossiers on formulae manufactured from protein hydrolysates submitted by food business operators for assessment by EFSA in the future. EFSA will be informed by the Commission by letter when the applicant has been asked by the Commission to transmit the dossier to EFSA for scientific assessment. 1.1.2 | Terms of Reference In accordance with Article 29 of Regulation (EC) No 178/2002,5 the European Commission requests the European Food Safety Authority to issue scientific opinions on infant and followon formula manufactured from protein hydrolysates in particular, depending on the nature of the application, on: 1Commission Directive 2006/141/EC of 22 December 2006 on infant formulae and followon formulae and amending Directive 1999/21/EC, OJ L 401, 30.12.2006, p. 1. 2OJ L 25, 2.2.2016, p. 1. 3Regulation (EU) No 609/2013 of the European Parliament and of the Council of 12 June 2013 on food intended for infants and young children, food for special medical purposes, and total diet replacement for weight control and repealing Council Directive 92/52/EEC, Commission Directives 96/8/EC, 1999/21/EC, 2006/125/EC and 2006/141/EC, Directive 2009/39/EC of the European Parliament and of the Council and Commission Regulations (EC) No 41/2009 and (EC) No 953/2009, OJ L 181, 29.6.2013, p. 35. 4EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2014. Scientific Opinion on the essential composition of infant and followon formulae. EFSA Journal 2014;12(7):3760. 5Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety, OJ L 31, 1.2.2002, p. 1. 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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 4 of 11 | NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE 1. the safety and suitability for use by infants of a specific formula manufactured from protein hydrolysates; If the formula under evaluation is considered to be safe and suitable for use by infants, the European Food Safety Authority is also asked to advise on the minimum specific criteria on protein source, protein processing and protein quality of the formula that need to be satisfied for the safety and suitability of such formulae to be demonstrated. 2. the product's efficacy in reducing the risk of developing allergy to milk proteins; 3. the product's efficacy in reducing the risk of developing allergy/allergic manifestations to allergens in general. 1.2 | Interpretation of the Terms of Reference For the particular dossier which is the subject of the present opinion, the request from the European Commission was related to the assessment of the nutritional safety and suitability of a protein hydrolysate for use in infant and followon formulae. The interpretation by the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) is that the safety of food enzymes or the combination of food enzymes that are used in the manufacture of the protein hydrolysate, is not to be assessed in this opinion. The assessment of the safety of the individual food enzymes is performed by the EFSA Panel on Food Enzymes (FEZ) according to the guidance and statements of the CEF/CEP Panel (EFSA CEF Panel,2009, 2016; EFSA CEP Panel,2019). Therefore, the conclusions of the Panel are related to the nutritional safety and suitability of the specific protein hydrolysate used to manufacture the infant and followon formulae for which the dossier has been submitted. They are not related to the safety of the protein hydrolysate in general, including the safety of the individual enzymes or their combination. Neither are they related to the safety of the final formula. This is justified as the composition of the formula with respect to substances other than the protein fraction should comply with the compositional requirements laid down in Commission Delegated Regulation (EU) 2016/1276 in order to ensure the nutritional safety and suitability for use by infants. The conclusions of the Panel also do not refer to the efficacy of the formula in reducing the risk of developing allergic manifestations. 2 | DATA AND METHODOLOGIES 2.1 | Data The assessment of the nutritional safety and suitability of the specific protein hydrolysate derived from sources of skimmed cow's milk (including nonfat) and whey protein concentrates and used in infant and followon formulae7 is based on the data supplied in the dossier submitted to EFSA and the additional information provided by the food business operator (FBO) upon request. A common and structured format for the presentation of dossiers related to infant and followon formulae manufactured from protein hydrolysates is described in the EFSA scientific and technical guidance for the preparation and presentation of an application for authorisation of an infant and/or followon formula manufactured from protein hydrolysates.8 As outlined in this guidance, it is the duty of the FBO who submitted the dossier to provide all available scientific data which are pertinent to the dossier. The procedure followed by EFSA for handling dossiers on formulae manufactured from protein hydrolysates, the various steps in the procedure and estimated timelines are described on EFSA's website.9 6Commission Delegated Regulation (EU) 2016/127 of 25 September 2015 supplementing Regulation (EU) No 609/2013 of the European Parliament and of the Council as regards the specific compositional and information requirements for infant formula and followon formula and as regards requirements on information relating to infant and young child feeding. OJ L 25, 2.2.2016, pp. 1–29. 7Infant formula means food intended for use by infants during the first months of life and satisfying by itself the nutritional requirements of such infants until the introduction of appropriate complementary feeding. Followon formula means food intended for use by infants when appropriate complementary feeding is introduced and which constitutes the principal liquid element in a progressively diversified diet of such infants. 8EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2017. Scientific and technical guidance for the preparation and presentation of an application for authorisation of an infant and/or followon formula manufactured from protein hydrolysates. EFSA Journal 2017;15(5):4779, 24 pp. https:// doi. org/ 10. 2903/j. efsa. 2017. 4779. 9http:// www. efsa. europa. eu/ sites/ defau lt/ files/ appli catio ns/ apdes kappl workf lownu triin fant. pdf. 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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 | 5 of 11 NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE 2.2 | Methodologies The assessment follows the methodology set out in the EFSA guidance for the preparation and presentation of an application for authorisation of an infant and/or followon formula manufactured from protein hydrolysates. Previous EFSA work10 and the regulatory framework11 were also taken into account. As the formula, in which the protein hydrolysate under evaluation is used, is marketed only in powder form, stability data were not evaluated for the formula (even though requested in the scientific and technical guidance8) as it is not expected that hydrolysis continues in powdered formulae. 3 | ASSESSMENT 3.1 | Characterisation of the protein hydrolysate The protein hydrolysate under evaluation is produced in two different production sites using different source materials and slightly different production procedures. Protein source: The protein hydrolysate under evaluation is produced from a mixture of sources of skimmed cow's milk (including nonfat) and whey protein concentrates (WPCs). Different combinations of these source materials are used in the production, resulting in distinct mixtures that are used in the manufacture of the protein hydrolysate. The resulting whey: casein (w/w) ratio is in all cases 60:40, with a content of total solids between around . Individual intact proteins in the sources of skimmed cow's milk (including nonfat) and whey protein sources have been identified by reversedphase high performance liquid chromatography with UV detection (RPHPLC–UV) in three to five batches of each source material. Their molecular masses were determined by RPHPLC–UV, quadrupole, time of flight mass spectrometry (RPHPLC–UVQTOF–MS) in one batch of each source material. Measurements were performed at the time of analysis by a nonaccredited external laboratory. A laboratory report, including the description of the methods used, and a validation report were provided. The FBO described that there are variations in the detailed composition of the source materials used as feedstock for the processes and this is to be expected of dairy products. They described that during the production process the starting materials are selected to provide the prespecified starting whey: casein ratio and the degree of hydrolysis is monitored. They also described that the main differences in the source materials used are nonprotein components such as minerals, and that this is addressed when developing the formulation of the final formula, i.e. by addition of other mineral ingredients to meet nutrient requirements and label claims of the finished products. The Panel notes that the protein composition is similar within the different analysed batches of each of the starting materials. Based on the information provided, with a detailed characterisation of the source materials and especially of the resulting protein hydrolysate from the two production sites, the Panel did not seek further information on specifications for the source materials used. Protein processing: The protein hydrolysate is produced under ISO 22000:2005, ISO/TS 220021:2009 and additional Food Safety System Certification (FSSC) 22,000 requirements in one production site according to a certificate provided and under Hazard Analysis and Critical Control Points (HACCP) and Good Manufacturing Practice (GMP) standards in the other facility, approved by the US Food and Drug Administration (FDA) for production of infant formula, according to a certificate provided. Slightly different protein processing is used in the two different production sites, which also use different combinations of source materials. At site 1, of WPC with a protein content is combined with to give a total source protein content This mixture is hydrated and heated for hydrolysis with an initial pH The , is added to the mixture in an amount of . The Panel considers that the EFSA question number under which the enzyme has been assessed for its safety is . Hydrolysis duration is . The enzyme is subsequently inactivated at applied for , followed by a heating to . The resulting protein hydrolysate is . At site 2, a combination of WPCs with protein contents of are combined with milk powder to give a total source protein content of . This mixture is hydrated and heated for hydrolysis to with an initial pH of is added in an amount of of substrate protein, 10EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2014. Scientific Opinion on the essential composition of infant and followon formulae. EFSA Journal 2014;12(7):3760, 106 pp. https:// doi. org/ 10. 2903/j. efsa. 2014. 3760. 11https:// ec. europa. eu/ food/ safety/ label ling_ nutri tion/ speci al_ groups_ food/ child ren_ en. 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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 6 of 11 | NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE equivalent to protein, to the mixture. Hydrolysis duration is . The enzyme is subsequently inactivated at applied for . The resulting protein hydrolysate is . According to certificates of analysis provided for three samples of protein hydrolysate powder from each of the two production sites, the residual protease activity was below the limit of quantification of the test method used . Taking the LOQ as the upperbound limit of residual activity, at least of the enzyme is inactivated. The method of analysis has been described and information on how the LOQ was derived was provided by the FBO. Degree of hydrolysis and molecular weight distribution of peptides, content of free amino acids and residual proteins: The degree of hydrolysis (DH) lies between . The FBO indicates that the calculation of DH is based on the formula DH[%] = h/htot × 100, where h (number of cleaved peptide bonds) is derived by measuring the increase in free amino groups in the source material. Free amino nitrogen (AN) is determined before and after hydrolysis by formol titration. Total nitrogen (TN) after hydrolysis is measured via Dumas combustion using an inhouse method that has been described. Analytical results for TN after hydrolysis were reported for 14 independently produced batches. htot (total number of peptide bonds) is derived from published data on the amino acid composition of the source material. Charts on the DH of all batches produced during 2015–2018 at site 1 and for the years 2012–2017 in site 2 were provided. A signed validation protocol was included in the dossier. An independent analysis of the 14 batches of protein hydrolysate was performed by an external laboratory using the ophthalaldehyde (OPA) method and this yielded higher results for DH, in the range of . These results and the method description were presented in a signed report of the external laboratory. No residual intact proteins were found at a concentration above the limit of detection for each of the 7 indicator proteins targeted for analysis by RPHPLC–UV (the same method as described above) in the 14 batches of protein hydrolysate. Results were presented in a signed report of the external laboratory. No free amino acids were reported to be present in quantifiable amounts in analyses of 14 batches of the protein hydrolysate using AOAC methods with modifications. Values for the molecular weight distribution of peptides were provided for 14 independently produced batches ( from production site 1 ( ) and from production site 2 ( )), for which a signed analysis report has been provided. The molecular weight distribution of peptides, based on these 14 batches, is on average in % w/w (range): 1–499 Da: 500–999 Da: 1000–1999 Da: 2000–2999 Da: 3000–4999 Da: > 5000 Da: The molecular weight distribution of peptides was measured by the analytical laboratory of the FBO by Size Exclusion Chromatography . Information on the column ( was provided as well as information on the calibration of the system with regards to molecular weight, including details on the calibrators used and data indicating the reproducibility of the method. Regarding Maillard reaction products, the concentrations of furosine, carboxymethyllysine (CML), εdeoxyfructosyllysine and blocked and total lysine for 14 batches of the spraydried protein hydrolysate were reported in a signed report of the external laboratory and compared with published data for other dried milk products, when available. Descriptions of the analytical methods used were provided. A statement of validity of the methods used has been provided. Data on the liquid hydrolysate have not been provided. The FBO states that Maillard reaction products in the liquid hydrolysate are expected to be lower, owing to the additional heat treatment step applied when spraydrying. The FBO indicates that furosine concentrations ( ) were generally within the range of values reported for spraydried skimmed milk (1000–5369 mg/kg protein; Erbersdobler etal.,2002; Van Renterghem & De Block,1996). CML concentrations were higher than those reported for spraydried milk powder or powdered cow's and goat's milk infant formula with intact protein (2–211 mg/kg protein; Prosser etal.(2019) and Erbersdobler etal.(2002)). CML concentrations were, however, within the range of those described for bovine whey protein hydrolysates (42–900 mg/kg protein; Prosser etal.,2019). Concentrations of blocked lysine were somewhat higher ( ) than those reported for spraydried milk powder (0%–7%; Erbersdobler & Hupe,1991; Nursten,2005 or spraydried infant formula 5%–10%; Finot,1983). The Panel notes that even if different starting materials and slightly different production processes were used in the different production sites, the molecular weight distribution of peptides, the concentrations of residual intact proteins (i.e. being all below the limit of detection) and the concentrations of Maillard reaction products are similar. The Panel considers that the protein hydrolysate that is used in the manufacture of the infant and followon formula for which the dossier has been submitted is sufficiently characterised with respect to the hydrolysed protein. 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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 | 7 of 11 NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE 3.2 | Characterisation of the formula manufactured from the protein hydrolysate used in the clinical studies provided The infant formula that is manufactured from the protein hydrolysate and that is used in the clinical study provided, complies with the compositional criteria of Regulation (EU) 2016/127,2 except for its content of vitamin D, which is in line with Directive 2006/141/EC.1 The protein content of this formula is 0.55 g/100 kJ (2.3 g/100 kcal). The amino acid profile meets the one laid down in Annex IIIA of Regulation (EU) 2016/127. The formula also contained a blend of . The infant formula, produced in powder form, is manufactured following GMP and the HACCP standards according to certificates provided. The Panel considers that the infant formula that is used in the pertinent human intervention study is sufficiently characterised. 3.3 | Safety and suitability of the infant and followon formula The FBO submitted an unpublished full study report(2024) of a doubleblind randomised controlled trial (RCT) conducted with 245 infants in the USA. Infants were randomised at the age of 10–14 days and were supposed to exclusively consume the study formulae up to the age of 120 days and thereafter, together with complementary foods, up to the age of 365 days. This assessment focussed on the time period from 10 to 14 days to 120 days of age. Randomisation was stratified by centre (total of 13 centres, however, 1 centre recruited around 40% of the infants) and sex. A total of 122 infants were allocated to consume the formula manufactured from hydrolysed protein for which the dossier has been submitted (hydrolysed formula, HF) and 123 infants a formula manufactured from intact cow's milk protein (control formula, CF). The study was designed as an equivalence study for the primary outcome of weight gain and an equivalence margin of ± 3 g/day was set a priori. For randomisation, centres were provided with sealed envelopes containing the product codes. Upon request by EFSA, the FBO explained that formula tins were but were otherwise identical. were used to identify the products dispensed in each group. Only personnel of the sponsor could break the blinding in case of a medical emergency, but this was not necessary during the study. The HF had a protein content of 0.55 g/100 kJ (2.3 g/100 kcal) and the CF of 0.45 g/100 kJ (1.9 g/100 kcal). The composition of both formulae complied with Regulation (EU) 2016/127, except for the protein source and its content of vitamin D, the latter being in line with Directive 2006/141/EC. Healthy term exclusively formulafed infants, 10–14 days of age, from singleton births with a birth weight of > 2500 g were included in the study. Infants were excluded if their weight at enrolment (V1) was < 98% of the birth weight. Largeforgestationalage infants of diabetic mothers were also excluded. The primary outcome of the study was the average weight gain from enrolment to 120 days of age. Secondary outcomes included, among others, length and head circumference (HC) gains, achieved body weight, length and HC, weightforage, lengthfor age and HCforage zscores, formula intake, adverse events, stool characteristics, tolerance of formula and quality of life. Infants were weighed naked once at each visit using a standard paediatric scale. Length was measured three times at each of the six visits in a recumbent position, one examiner holding the head against a fixed headboard and another examiner positioning the feet at the movable footboard. HC was measured three times, using a nonstretchable cloth or vinyl tape. The largest values of the three measurements of length and HC were taken. Upon request from EFSA, the FBO explained that it was decided to include the largest of the three measurements in the analysis, because it was considered that actual length and HC was more likely to be underestimated rather than overestimated. All values were checked for significant deviations between the values obtained from the three measurements. Formula intake, stool characteristics and tolerance of the formula were assessed using 24h recalls at each of the five visits from V2 onwards. Baseline stool characteristics and tolerance of the formula were also elicited using 24h recalls at V1. Quality of life was evaluated using the Pediatric Quality of Life Inventory™ using the Family Impact Module (PedsQL FIM)- Acute and the Infant Scales (PedsQL Infant Scales). Adverse events were recorded only when they had been confirmed medically. Feeding of the intervention formulae started at the first feeding after completion of V1 (10–14 days of age). Anthropometric measures were taken at the age of 10–14 (V1), 30 (V2), 42 (V3), 60 (V4), 90 (V5) and 120 (V6) days. Stool characteristics and tolerance of the formula were also assessed at these visits. Formula intake was evaluated from V2 onwards. The PedsQL FIM was administered also from V2 onwards while the PedsQL Infant Scales were run at V6. At each visit, empty formula tins were collected and caregivers were asked about the intake of formula other than study formulae and intake of complementary foods. Criteria for withdrawing infants from the study were set a priori and included, among others, any breastmilk consumption after randomisation, discontinuation of study formula consumption or drop in study formula intake ≥ 50% for ≥ 5 consecutive days, failure to comply with scheduled study windows, closure of a study site or loss to follow up. Analyses were performed in a population set which included infants who had available outcome data both at enrolment and at V6 and who had not been withdrawn from the study. This population set was denominated by the FBO as full 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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 8 of 11 | NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE analysis set (FAS). In addition, analyses were presented in a population set in which additionally infants who had not strictly met the inclusion or exclusion criteria of the study were excluded. This concerned an additional seven infants, three in the CF and four in the HF group. This population set was denominated by the FBO as per protocol set (PPS). The analyses in the ‘PPS’ were only done for anthropometric measurements. The Panel notes that the definitions of the ‘FAS’ and the ‘PPS’ used by the FBO deviate from the commonly used definitions of the FAS and the PPS. Upon request from EFSA, the FBO also provided an analysis on weight gain between V1 and V6 based on the ‘PPS’ but excluding infants who had consumed any nonstudy formula or complementary foods between V1 and V6, an analysis excluding infants whose measurements were taken outside the prespecified visit windows as well as an analysis excluding infants who had consumed nonstudy formula or complementary foods and/or had measurements taken outside the visit windows. Analysis of variance (ANOVA) was used in the statistical analyses which included terms for sex and study group. Equivalence for the primary outcome was tested using the twoonesidedtests (TOST) procedure. zScores of anthropometric data were calculated using WHO child growth standards (WHO,2006; WHO Multicentre Growth Reference Study Group,2006). Sample size was calculated assuming a difference in weight gain between groups of 0.5 g/day and a standard deviation (SD) of 6 g/day, at a power of 80%, a significance level of 5% and using an equivalence margin of 3 g/day. It was determined that 78 infants per group were needed. From the originally randomised 245 infants (123 to the CF group and 122 to the HF group), one infant in the CF group did not start the study. The primary analysis of weight gain included in the ‘FAS’ 91 infants in the CF group (74% of the originally enrolled infants) and 84 infants in the HF group (67% of the originally enrolled infants). The analysis in the ‘PPS’ included 89 infants in the CF group and 81 in the HF group. In the additional analyses provided by the FBO upon request, infants in the CF group and infants in the HF group were included after excluding those who had consumed any nonstudy formula or complementary foods. When excluding infants who had measurements taken outside the study window, infants in the CF group and infants in the HF group were included. Finally, when excluding infants meeting both conditions, infants in the CF group and infants in the HF group were included. Baseline characteristics of originally randomised infants in the HF and CF groups were similar. In the ‘FAS’ analysis, the mean difference in weight gain from 14 to 120 days of age was 0.49 g/day (90% confidence interval (CI) −1.10 to 2.08 g/day). In the ‘PPS’ analysis, the mean difference was 0.28 g/day (90% CI −1.33 to 1.89 g/day). The results provided by the FBO upon request from EFSA excluding infants who had consumed nonstudy formula and/or complementary foods and/or had study visits outside the predefined time windows showed similar results with the 90% CI remaining within the prespecified margin of equivalence. There was a significant interaction between study group and gender, with females in the HF group showing on average higher weight gain than females in the CF group (on average 3.1 g/day) and males in the HF group showing a lower weight gain (on average −2.0 g/day). Formula intake was not different between boys and girls. The Panel notes that the differences in weight gain originated from differences in weight gain between boys and girls in the control group and not from differences in weight gain of boys and girls in the group consuming the intervention formula. Moreover, average weight gain in boys and girls in both groups fell between the 50th and the 75th percentile of the WHO child growth standards. Length and HC gains were similar, with no interaction between study group and gender. The analyses of achieved weight, length and HC as well as variables expressed as zscores at V6 were consistent with the results of the analysis on growth. Formula intake between groups was similar throughout the study. At day 120, infants consumed a mean ± standard error (SE) formula volume of 1029 ± 35.5 mL in the CF group and 1035 ± 38.4 mL in the HF group. Discontinuation of the study owing to intolerance of the formula within the whole study period of 365 days occurred in seven infants in each group. Hours of crying, number of spitups, difficulty of napping, number of nightwakings, night time sleep quality, stool frequency, stool consistency, fussiness and gassiness were similar between groups. At day 120, none of the subscales of the PedsQL FIMAcute and the PedsQL Infant Scales showed a statistically significant difference between groups. The incidence of adverse events clustered by body system occurring throughout the whole study period of 365 days was not significantly different between groups as was the incidence of specific adverse events and the number of participants in whom at least one adverse event occurred. None of the serious adverse events which occurred were judged by the investigators to be related to the study products. The Panel considers that this study shows that an infant formula manufactured from the protein hydrolysate described in Section3.1 with a protein content of 0.55 g/100 kJ (2.3 g/100 kcal) and consumed as the sole source of nutrition for 3 months leads to growth that is similar to an infant formula manufactured from intact cow's milk protein with a protein content of 0.45 g/100 kJ (1.9 g/100 kcal). The Panel concludes that the protein hydrolysate under evaluation is a nutritionally safe and suitable protein source for use in infant formula, as long as the infant formula in which it is used contains a minimum of 0.55 g/100 kJ (2.3 g/100 kcal) protein and complies with the compositional criteria of Commission Delegated Regulation (EU) 2016/127 and the amino acid pattern in Annex IIIA of the Regulation. No experimental data have been provided on the nutritional safety and suitability of this protein source in followon formula. However, given the fact that followon formula is consumed in conjunction with complementary foods and the protein source is considered nutritionally safe and suitable in an infant formula that can be the sole source of nutrition of 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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 | 9 of 11 NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE young infants, the Panel considers that the protein hydrolysate under evaluation is also a nutritionally safe and suitable protein source for use in followon formula, as long as the followon formula in which it is used contains a minimum of 0.55 g/100 kJ (2.3 g/100 kcal) protein and complies with the compositional criteria of Commission Delegated Regulation (EU) 2016/127 and the amino acid pattern in Annex IIIA of the Regulation. 3.4 | Uncertainties related to the nutritional safety and suitability of the infant formula The Panel notes that the definitions of the FAS and the PPS used by the FBO deviate from the commonly used definitions of the FAS and the PPS. However, the Panel considers that the posthoc analyses provided by the FBO upon EFSA's request, which showed results similar to those in the FAS and PPS (as defined by the FBO), increase confidence in the study's findings. The Panel also notes that weight gain in boys in the CF group was lower than in girls when standardised for age and sex, while both boys and girls in the HF group followed the same growth trajectories. This resulted in boys in the CF group showing lower weight gain compared to boys in the HF group, and girls in the CF group showing higher weight gain compared to girls in the HF group. However, their weight gain was within the range expected according to WHO child growth standards. There were no relevant differences in formula intakes between boys and girls, indicating that differences in formula intake could not explain the observed differences in growth between boys and girls in the CF group. 4 | CONCLUSIONS The Panel concludes that: • the protein hydrolysate for which the dossier has been submitted and that is to be used in the manufacture of infant and followon formulae is sufficiently characterised with respect to the hydrolysed protein; • the minimum specific criteria for characterisation of the protein hydrolysate with respect to the protein source, protein processing and protein quality, as requested in the terms of reference, are those given in Section3.1; • the protein hydrolysate for which the dossier has been submitted is a nutritionally safe and suitable protein source for use in infant and followon formulae, as long as the formula in which it is used contains a minimum of 0.55 g/100 kJ (2.3 g/100 kcal) protein and complies with the compositional criteria of Commission Delegated Regulation (EU) 2016/127 and the amino acid pattern in Annex IIIA of the Regulation. 5 | DOCUMENTATION AS PROVIDED TO EFSA Application for authorisation of a formula manufactured from protein hydrolysates. August 2019. Submitted by Healthcare Reckitt B.V. 6 | STEPS TAKEN BY EFSA 1. The technical dossier was received by EFSA on 16/04/2019. 2. A letter from the European Commission with the request for a scientific opinion on the safety and suitability for use by infants of an infant and followon formula manufactured from protein hydrolysate was received by EFSA on 16/04/2019. 3. The scientific evaluation procedure started on 09/09/2019. 4. On 30/09/2019, the Working Group on protein hydrolysatebased formula of the NDA Panel agreed on a list of questions for the FBO to provide additional information to accompany the dossier. The scientific evaluation was suspended on 11/10/2019 and was restarted on 26/07/2024. 5. On 18/09/2024, the Working Group on protein hydrolysatebased formula of the NDA Panel agreed on a list of questions for the FBO to provide additional information to accompany the dossier. The scientific evaluation was suspended on 01/10/2024 and was restarted on 16/12/2024. 6. During its meeting on 2930/01/2025, the NDA Panel, having evaluated the data, adopted an opinion on 29/01/2025. ABBREVIATIONS % w/w percentage on a weightperweight basis AN amino nitrogen ANOVA analysis of variance AOAC Association of Analytical Communities CEF Panel Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids CEP Panel Panel on Food Contact Materials, Enzymes and Processing Aids CF control formula 18314732, 2025, 3, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9278 by Readcube (Labtiva Inc.), Wiley Online Library on [09/06/2025]. 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