Nutritional safety and suitability of a specific protein hydrolysate manufactured by Fonterra Co-operative Group Ltd derived from a whey protein concentrate and used in infant formula and follow-on formula

EFSA Journal. 2025;23:e9160. | 1 of 11 https://doi.org/10.2903/j.efsa.2025.9160 efsa.onlinelibrary.wiley.com/journal/1831-4732 SCIENTIFIC OPINION Nutritional safety and suitability of a specific protein hydrolysate manufactured by Fonterra Cooperative Group Ltd derived from a whey protein concentrate and used in infant formula and followon formula 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: 28 November 2024 DOI: 10.2903/j.efsa.2025.9160 Abstract The European Commission asked EFSA to deliver an opinion on the nutritional safety and suitability for use in infant and followon formula of a specific protein hydrolysate from whey protein concentrate manufactured by Fonterra Cooperative Group Ltd. Fonterra Cooperative Group Ltd. submitted a dossier to the European Commission to request an amendment of Regulation (EU) 2016/127 with respect to the protein sources that may be used 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 2.0 g/100 kcal and consumed as the sole source of nutrition by infants for 3 months led to a growth similar to a formula manufactured from intact cow's milk with the same protein content. Data on gastrointestinal 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 is 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 2.0 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..............................................................................................................................................................................................4 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 ..........................................................................................................................................................................................................6 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 5.1. 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........................................................................................................................................................................................................................ 10 18314732, 2025, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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: 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. 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, 12(7), 3760. https:// doi. org/ 10. 2903/j. efsa. 2014. 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, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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 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 formula. The interpretation by the Panel on Nutrition, Novel Foods and Food Allergens (NDA) is that the safety of food enzymes or their combination 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 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). The assessment of food enzymes is ongoing at the time of the adoption of the present opinion. 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 formula 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) 2016/127 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 whey protein concentrate and used in infant formula6 and followon formula7 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 from EFSA. 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 online.9 In accordance with Article 38 of Regulation (EC) No 178/2002 and taking into account the protection of confidential information and of personal data in accordance with Articles 39, 39a to 39e of the same Regulation and of the Decision of the EFSA's Executive Director laying down practical arrangements concerning transparency and confidentiality,10 the nonconfidential versions of the data submitted by Fonterra are published in OpenEFSA.11 According to Art. 32c (2) of Regulation (EC) No 178/2002 and according to the Decision of EFSA's Executive Director laying down the practical arrangements on the presubmission phase and public consultations, EFSA carried out a public consultation (PC1171)12 from 17 October to 7 November 2024 on the nonconfidential version of the submission by Fonterra. 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,8 as well as in the explanatory note to the 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 (with a view to amend 6Infant 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. 7Followon 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). (2021). Scientific and technical guidance for the preparation and presentation of a dossier for evaluation of an infant and/or followon formula manufactured from protein hydrolysates (Revision 1). EFSA Journal, 19(3), 6556. https:// doi. org/ 10. 2903/j. efsa. 2021. 6556. 9https:// www. efsa. europa. eu/ sites/ defau lt/ files/ appli catio ns/ apdes kappl workf lownu triin fant. pdf. 10Practical arrangements concerning the Transparency Regulation are accessible under: https:// www. efsa. europa. eu/ en/ corpo ratepubs/ trans paren cyregul ationpract icalarran gements. 11https:// open. efsa. europa. eu/ quest ions/ EFSA-Q202100339 . 12https:// conne ct. efsa. europa. eu/ RM/s/ consu ltati ons/ publi ccons ultat ion2/ a0lTk 00000 2dKqX/ pc1171. 18314732, 2025, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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 Regulation (EU) 2016/127).13 A previous EFSA opinion of 201414 and the regulatory framework15 were also taken into account. As the infant 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 Protein source The protein hydrolysate under evaluation is produced from whey protein concentrate (WPC) from cow's milk with an average protein content of 16 an average lactose content of and an average fat content of . Raw material specifications and certificates of analysis were provided. Individual intact proteins in the source material have been identified by reversed phasehigh performance liquid chromatography (RPHPLC) based on the method by Elgar etal.(2000). Protein processing The protein hydrolysate is produced under Hazard Analysis and Critical Control Points (HACCP) and additional Food Safety System Certification (FSSC) 22000 requirements. Certificates of FSSC 22000 certifications were provided. In order to produce the hydrolysate, the source material (WPC) is hydrated to a solids content of and heated to for the enzymatic hydrolysis step that is performed . This step is followed by a heat treatment of . The enzymatic hydrolysis step is carried out at and followed by a heat treatment of . The food enzymes used for hydrolysis have been identified by the FBO. the food enzymes employed in the process are currently under safety assessment by the EFSA CEP Panel, while for the assessment has been finalised In the hydrolysis step, a serine endopeptidases is used. The hydrolysis step is performed with The FBO states that the final heat treatment step reduces the enzyme activity by . Degree of hydrolysis and molecular weight distribution of peptides, content of free amino acids and residual proteins Descriptions of all analytical methods used were provided together with documentation on method validation and accreditation of the laboratories performing the analyses, except for the analyses of free amino acids which were based on the method described by Mondino etal.(1972), for which the validation documentation was not available from the laboratory performing the measurements. The degree of hydrolysis (DH) was approximated by dividing the free amino nitrogen (AN) in the protein hydrolysate, analysed by a validated ophthaldialdehyde (OPA) method, by the total number of peptide bonds in the source material, htot, as reported by AdlerNissen(1986) for whey protein. The FBO presents this ratio as DH. The Panel notes that the data presented by the FBO are not equivalent to DH and are only considered to approximate DH (as explained in 13https:// www. efsa. europa. eu/ en/ suppo rting/ pub/ en1858. 14EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2014. Scientific Opinion on the essential composition of infant and followon formulae. EFSA Journal, 2(7), 3760. https:// doi. org/ 10. 2903/j. efsa. 2014. 3760. 15https:// ec. europa. eu/ food/ safety/ label ling_ nutri tion/ speci al_ groups_ food/ child ren_ en. 16Calculated as Nx6.38. 18314732, 2025, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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 EFSA(2020)). The average of this ratio, expressed as a percentage, based on 18 samples from five independently produced batches was with a standard deviation (SD) of . The manufacturing specifications of the FBO for this ratio is . Certificates of analyses were presented. The residual amount of proteins was analysed in three ways: First, the FBO quantified the amount of residual in the protein hydrolysate by using an enzymelinked immunosorbent assay (ELISA, five samples, batchtobatch variability and certificates of analyses provided). The detected by ELISA was on average per kg powder. The FBO explained that the ELISA method also detects epitopes presenting peptides together with intact and therefore likely overestimates the content of intact . The FBO used this method to estimate the content of residual proteins in the protein hydrolysate and to set a limit of for product release. Then, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) was used to qualitatively assess the presence of residual proteins, but no proteins were detected. Finally, residual intact and were quantified using reverse phasehigh performance liquid chromatography–mass spectrometry (RPHPLCMS; validated). The three proteins were below the limit of detection of the method of protein hydrolysate, corresponding to a reduction of of the original protein content and in agreement with the ELISA results. The average amount of free amino acids in the protein hydrolysate (powder form) is ( ). Data on batchtobatch variability of 18 samples of five independently produced batches have been presented together with the certificates of analyses. Average values for the molecular weight distribution of peptides and data on the batchtobatch variability of 18 samples from five batches were presented together with the certificates of analyses. Manufacturing specification limits as minimum and maximum percentage for each molecular weight range are given in brackets. < 1000 Da: 1000–5000 Da: 5000–20,000 Da: > 20,000 Da: The molecular weight distribution of peptides was measured by size exclusionHPLC (SEHPLC) with UV detection . Information on the column ( ) used was provided. 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 were provided. Concentrations of furosine in 18 samples of five batches of the protein hydrolysate (certificates of analyses provided) were analysed using a validated method based on the Agriculture Industry Standard of the People's Republic of China NY/ IT 9392016. Furosine concentrations ranged from which was in the range of furosine concentrations, for example, in an ultrahightemperature (UHT) milk analysed as a control test sample. The Panel considers that the protein hydrolysate that is used in the manufacture of the infant and followon formulae for which the dossier has been submitted is sufficiently characterised with respect to the hydrolysed protein. 3.2 | Characterisation of the formula manufactured from the protein hydrolysate used in the clinical studies provided The infant formula manufactured with the protein hydrolysate that is used in the clinical studies submitted complies with the compositional criteria of Regulation (EU) 2016/127, except for its content of vitamin D, which is 0.62 μg/100 kJ (2.6 μg/100 kcal) and higher than the maximum content of 0.60 μg/100 kJ (2.5 μg/100 kcal) set up in Regulation (EU) 2016/127. The protein content of this formula is 0.48 g/100 kJ (2.0 g/100 kcal) and is based on a calculation of total nitrogen × 6.25. are added as free amino acids to the formula. The amino acid profile meets the one laid down in Annex IIIA of Regulation (EU) 2016/127, except for the concentrations of which is and lower than the minimum content of laid down in Regulation (EU) 2016/127 and the concentration of which is and lower than the minimum of ) laid down in Regulation (EU) 2016/127. Certificates of analyses of two batches of the infant formula were provided by the FBO. The FBO states that . Analyses were carried out in the laboratory of the manufacturer or in an ISO 17025 accredited laboratory. The infant formula, produced in powder form, is manufactured following FSSC 22000 standards, according to certificates provided. The two batches of the formula were analysed for their furosine and carboxymethyllysine (CML) concentrations (certificates of analyses provided). The methods used for analyses were validated and descriptions were provided. Furosine was analysed using , and CML analyses were done using . In the two batches, concentrations of furosine were ; CML concentrations were . The control formula used in the study described in Section3.3 had a furosine concentration of and a CML concentration of in both 18314732, 2025, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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 batches. The concentrations were within the range of values reported for infant formula based on a database maintained by the Technical University Dresden as reported by the FBO. 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 presented an unpublished full study report (Gunn etal.,2023) of one pertinent doubleblind randomised controlled trial (RCT) with 249 infants who were randomised in blocks of 10, stratified by site and sex, to consume exclusively either the infant formula manufactured from hydrolysed whey protein for which the dossier has been submitted (HF) or an infant formula manufactured from intact cow's milk protein (control formula, CF). An exclusively breastfed reference group of 50 infants was also included. This group was enrolled in a ratio of 4:1 in each study site and had similar baseline characteristics, including age at enrolment, as the formulafed groups. 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. The HF and the CF had protein contents of 0.48 g/100 kJ (2.0 g/100 kcal). The composition of both formulae complied with Commission Delegated Regulation (EU) 2016/127, except for the protein source of the HF. The study was carried out in Bulgaria and Germany. Healthy term exclusively formulafed infants up to 28 days of age with a birthweight between 2500 and 4500 g and a 5min Apgar score of ≥ 7 were included in the study. The randomisation schedule was computergenerated and provided to study sites in sealed opaque consecutively numbered envelopes with the indication whether it was for a male or female. The Panel notes the limitations of this approach in terms of successful blinding of the study. Formulae powders with identical smell and appearance were provided in metallic tins labelled with the unique code. The primary outcome of the study was the average weight gain from enrolment to day 90 of the study. Secondary outcomes included body weight, length, head circumference (HC), weightforage, weightforlength, lengthfor age zscores, adverse events and serious adverse events, stool consistency assessed by the Bristol stool form scale (Chumpitazi etal.,2010), stool frequency, formula intake, spitting up and regurgitation, flatulence and bloating, vomiting, crying/screaming episodes, colic, fussiness and sleep time. All anthropometric measurements at baseline were performed at the study sites (17 sites in Germany and three in Bulgaria had active participants) but during the COVID19 pandemic, some parents carried out measurements at home. At the final visit, parental home measurements were completed for out of infants (including all three feeding groups, including the breastfed reference group) in Bulgaria. In Germany, infants were measured by parents, three by health professionals in their offices and at the study sites (total number of infants measured ). For home measurements, parents were supplied with medical grade scales, measuring boards and tape measures for HC measurements. Parents received written instructions. In Bulgaria, telephone assistance was also available. The Panel assumes that home measurements were equally distributed among groups, because of the randomised nature of the study, and notes that in Germany 20% of all measurements at the final time point were done by parents, while it was 6% in Bulgaria. Intervention formula feeding began the evening after the initial enrolment visit (V1) for infants aged 0 to 28 days. The following measurements took place at days 30 (V2), 60 (V3) and 90 (V4) after the start of feeding the intervention formulae. Another study visit took place at the age 6 months (V5), during which also anthropometric parameters and tolerance to the product were assessed. Three days before each visit, parents were asked to record on diary cards the frequency and amount of formula fed per day and whether any complementary food had been consumed. Compliance was assessed by collecting the empty tins and by checking the diary cards. Analyses were performed in the intentiontotreat population (ITT, i.e. all individuals randomised) and in the per protocol population (PP, all individuals attending all visits with > 66.6% compliance and no major protocol violations, not having been given complementary foods and not having been fed more than one bottle of a formula other than the intervention formula during the whole study period). The ITT population was the population used for the primary analysis. For analysing the difference in weight gain over 90 days between intervention and control group an analysis of covariance (ANCOVA) was used with intervention and country and their interaction as fixed factors, gender as fixed blocking factor and gestational age at birth and baseline body weight as covariates. The Panel notes that, for the analysis of this primary endpoint, it is unclear how missing data were handled in the ITT analysis. The place at which measurements took place (study sites vs. at home) was initially included in the statistical model, but as there was no interaction with the formula groups it was removed from the final model. Continuous variables of secondary endpoints were analysed using a mixed model for repeated measures including all five measurement time points. An autoregressive covariance pattern was chosen. Posthoc comparisons were performed using the Tukey test for pairwise comparisons. For secondary endpoints, which were only assessed once after baseline, the same model as for the primary analysis was used. For categorical variables the chisquared test was applied. A Poisson 18314732, 2025, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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 model was used to assess incidence rates. zScores were calculated using WHO reference standards.17 The BenjaminiHochberg procedure was used to control for the false discovery rate and the pvalue for statistical significance for secondary outcomes was set at ≤ 0.0025. Sample size calculations were based on a twosided, twosample ttest assuming a standard deviation (SD) of 5–6 g/day and a difference in weight gain of 3 g/day. For having a 90% power at a 5% significance level, 86 infants per group were to be included. Assuming a 40% dropout rate, 143 infants per group were needed. In total, 249 infants were randomised, 123 to the HF group and 126 to the CF group. In the HF group, 56 discontinued the intervention and/or did not comply with the protocol up to V4 (45.5% of randomised infants): 11 were lost to follow up, 4 withdrew consent, 17 left the study because of intolerance and 24 did not comply or had a protocol violation. In the CF group, 50 stopped participation and/or did not comply with the protocol up to V4 (39.7%): 1 infant did not start the intervention, 12 were lost to follow up, 1 withdrew consent, 20 stopped because of intolerance and 16 did not comply or had a protocol violation. Hence, at V4, 67 infants in the HF and 76 infants in the CF group were in the PP population. At V5, the numbers were 59 and 68, respectively. From the data provided in response to a request for additional information from EFSA, the Panel notes that infants in the HF group ( of all randomised infants) and infants in the CF group ( of all randomised infants) had outcome data both at V1 and V4. The Panel notes the high dropout rate which was similar between both groups. Baseline characteristics of infants in the HF and CF groups were similar in both the ITT and PP populations. In the PP population, age and weight at inclusion in the HF group were mean ± SD 9.1 ± 8.3 days and 3304 ± 457 g (unadjusted) and in the CF group 9.3 ± 9.2 days and 3340 ± 502 g, respectively. However, age at baseline was considerably different between infants recruited in Bulgaria and those recruited in Germany. In Bulgaria, infants in the HF group had a mean (SD) age at baseline of 14.8 (8.21) days and those in the CF group 16.0 (9.14) days. In Germany, they were 3.5 (2.60) and 3.4 (3.18) days old, respectively. Including age instead of weight at baseline as covariate did not change the results of the analysis (analysis provided by the FBO upon request from EFSA). The mean difference in weight gain between V1 and V4 between the HF and the CF group was 1.9 g/day (95% CI 0.0 to 3.9 g/day) in the ITT and 2.4 g/day (0.3 to 4.5 g/day) in the PP population. The Panel notes that the upper bound of the 95% CI exceeded the predefined margin of equivalence. However, the Panel also notes that this likely reflected the lower weight gain in infants in the Bulgarian CF group compared to German infants in both feeding groups and Bulgarian infants in the HF group, a difference for which no explanation could be found. In addition, the Panel notes that both formulafed groups gained weight within the range expected according to WHO standards. Results for length, HC and anthropometric measures given as zscores were consistent with the conclusions for weight gain. Formula feeding volume was similar between groups. Formula intake was on average (95% CI) 828 mL (786 to 871 mL) in the HF group and 798 mL (758 to 837 mL) in the CF group at V4. In a repeated measures analysis, also including V5, there was no statistically significant treatment per time interaction. There were 111 adverse events reported in 123 infants (ITT population) in the HF group and 152 events in 125 infants in the CF group. At least one adverse event occurred in 53% of infants in the HF group and 60% in the CF group. They were judged by the investigators to be probably related to the study formula in 7 cases for the HF group and 12 cases for the CF group. All adverse events classified as serious were considered by the investigators as unrelated to the study formula. Stool frequency was lower in the HF group than the CF group [mean (95% CI) HF vs. CF: 1.42 (1.21–1.53) vs. 1.60 (1.40–1.80) bowel movements per day, which showed a statistically significant treatment per time interaction (p = 0.04) in a repeatedmeasures analysis]. Hard stools occurred in one infant in the HF and two infants in the CF group at V2 but not at subsequent visits. None of the other secondary tolerance outcomes were significantly different between groups, except for the number of screaming episodes which was higher in the CF (1%–2% of infants, depending on the visit) than the HF group (0% of infants). The Panel considers that the abovementioned differences in stool frequency and screaming episodes are not clinically significant. 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 0.48 g/100 kJ (2.0 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 the same protein content. 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 0.48 g/100 kJ (2.0 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 is the sole source of nutrition of 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 0.48 g/100 kJ (2.0 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. 17Using the SAS macros published by WHO (https:// www. who. int/ tools/ childgrowthstand ards/ software). 18314732, 2025, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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 3.4 | Uncertainties related to the nutritional safety and suitability of the infant formula The Panel notes that weight gain in Bulgarian infants in the CF group was lower than in the HF group and also lower than in German infants in the CF and HF groups and the breastfed reference group, even though their weight gain was within the range expected according to WHO reference standards. There were no relevant differences in baseline characteristics of infants or formula intakes between groups of formulafed infants. The same two batches of the control formula were used both in Bulgaria and Germany and batches did not differ in their composition as confirmed by certificates of analyses, so that differences in formula composition could not explain the differences in growth. 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 formula is sufficiently characterised with respect to its fraction of 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 a suitable protein source for use in infant and followon formula, as long as the formula in which it is used contains a minimum of 0.48 g/100 kJ (2.0 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 Dossier on the safety and suitability for use by infants of an infant formula manufactured from protein hydrolysates. September 2023. Submitted by Fonterra Cooperative Group Limited. 5.1 | Steps taken by EFSA 1. The technical dossier was received by EFSA on 15/06/2021. 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 06/09/2021. 3. The scientific evaluation procedure started on 15/09/2023. 4. On 13/11/2023, the Working Group on protein hydrolysatebased formula of the NDA Panel agreed on a list of questions for the food business operator to provide additional information to accompany the dossier. The scientific evaluation was suspended on 21/11/2023 and was restarted on 27/03/2024. 5. On 31/05/2024, the Working Group on protein hydrolysatebased formula of the NDA Panel agreed on a list of questions for the food business operator to provide additional information to accompany the dossier. The scientific evaluation was suspended on 04/06/2024 and was restarted on 04/07/2024. 6. During its meeting on 28/11/2024, the NDA Panel, having evaluated the data, adopted an opinion on 28/11/2024. ABBREVIATIONS AN amino nitrogen ANCOVA analysis of covariance CEP Panel on Food Contact Materials, Enzymes and Processing Aids CEF Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids CF control formula CI confidence interval CML carboxymethyllysine COVID19 Coronavirus disease of 2019 Da dalton DH degree of hydrolysis EC Enzyme Commission ELISA enzymelinked immunosorbent assay FBO Food business operator FEZ Panel on Food Enzymes FSSC Food Safety System Certification GMO genetically modified organism 18314732, 2025, 1, Downloaded from https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9160 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