Early walnut powder introduction for prevention of walnut sensitisation in high-risk infants with eczema; an open-label, randomized, non-inferiority trial

WISH-J study version 1.5 4/23/2025 1 / 37 Early walnut powder introduction for prevention of walnut sensitization in high-risk infants with eczema. An open-label, randomized, non-inferiority trial research plan Abbreviated name: Walnut sensitization inhibition test WISH-J Study Walnut Intake for Suppression of High-Level Sensitization in Japan Principal Investigator: Kiwako Yamamoto Physician, Department of Allergy, Allergy Center, National Center for Child Health and Development 〒2-10-1, Okura, Setagaya-ku, Tokyo 157-8535 Phone: 03-3416-0181 fax: 03-3415-9260 E-mail: [email protected] Research Office Representative: Mayako Saito Physician, Department of Allergy, Allergy Center, National Center for Child Health and Development 〒2-10-1, Okura 2-chome, Setagaya-ku, Tokyo 157-8535 Phone: 03-3416-0611 fax: 03-3415-9260 E-mail: allergy_resear[email protected] WISH-J study version 1.5 4/23/2025 2 / 37 Establishment and Revision History Version Date of amendment Implemented by Details 1.0 13/6/2024 Kiwako Yamamoto First version 1.1 2024/6/24. Kiwako Yamamoto Basis for setting the protein content of walnuts added in response to the technical expert's evaluation report. 1.2 2024/7/8 Kiwako Yamamoto Paragraph 1.8 deleted in response to points raised by the Preliminary Examination Subcommittee. 1.3 27/7/2024 Kiwako Yamamoto CRB Review Findings, Change description from 3 days per week to at least 3 days per week Change the way test meals are handed over. Possible disadvantages in the consent form added. 14.Corrected omission of research period January → November. 1.4 28/10/2024 Kiwako Yamamoto Change in total quantity of walnut powder. 1.5 4/23/2025 Kiwako Yamamoto Corrected discrepancies between research protocol and consent form WISH-J study version 1.5 4/23/2025 1 Table of Contents 1. Key contacts. ........................................................................................................................... 6 1.1. Principal investigator. ........................................................................................................... 6 1.2. Participating institutions and principal investigators 1.3.Research collaborator ............................................................................................................ 6 1.4.Research administration and coordination and management staff ........................................ 6 1.5. Statistician ............................................................................................................................ 6 1.6.Assignment and allocation..................................................................................................... 6 1.7.Data Management .................................................................................................................. 6 1.8. monitoring ............................................................................................................................ 6 1.9. chief auditor .......................................................................................................................... 6 1.10. Walnut Powder Provider .................................................................................................... 6 2.Background and rationale ......................................................................................................... 7 2.1.background ............................................................................................................................ 7 2.2.Overview of medicinal products, etc ................................................................................... 12 3.Matters related to the purpose of clinical research ............................................................... 13 3.1.Trial Phase ........................................................................................................................... 13 3.2.Sample size .......................................................................................................................... 13 3.3.Study Design........................................................................................................................ 14 Criteria for selection and exclusion of subjects for clinical research and discontinuation of clinical research ........................................................................................................................... 15 3.4.Subject inclusion Criteria .................................................................................................... 15 3.5.Exclusion Criteria ............................................................................................................... 15 3.6 Study intervention discontinuation and participant discontinuation/withdrawal........................................................20 3.7 Study discontinuation criteria....................................................... .20 4.Matters relating to the treatment of clinical research subjects. .......................................... 16 4.1.Subject Registration Procedures .......................................................................................... 16 4.2.Allocation Method and Allocation Adjustment Factors ...................................................... 17 4.3.Blinding ............................................................................................................................... 17 4.4.Emergency unblinding ......................................................................................................... 17 4.5.Study intervention................................................................................................................ 17 4.6.Observations/test items and treatment information to be reported ...................................... 20 5.evaluation item .......................................................................................................................... 24 5.1.Primary Outcome ................................................................................................................. 24 5.2 Secondary Outcome ............................................................................................................. 24 6.Assessment and reporting of adverse events .......................................................................... 25 6.1.Definition of adverse events ................................................................................................ 25 6.2.Serious adverse events ......................................................................................................... 25 6.3.Definition of non-serious adverse events ............................................................................ 26 6.4.Severity of adverse events ................................................................................................... 26 6.5.Adverse Event Reporting ..................................................................................................... 26 6.6.Reporting of diseases and other illnesses and response to outbreaks of serious diseases ... 26 7.Matters related to statistical analysis ..................................................................................... 27 7.1.Analysis target population .................................................................................................. 27 7.2.significance level ................................................................................................................. 27 7.3.Analysis Method .................................................................................................................. 27 7.3.1. Summary of Background Information ...................................................................... 27 7.3.2. Analysis of primary outcomes .................................................................................. 27 7.3.3. Analysis of secondary outcomes .............................................................................. 28 7.3.4. Analysis of safety outcomes ..................................................................................... 28 WISH-J study version 1.5 4/23/2025 2 7.4.Interim Analysis .................................................................................................................. 28 7.5.Procedure for changing the statistical analysis plan ............................................................ 28 8.Matters relating to access to original documents and other materials ........................................ 28 8.1.Scope of source documents ................................................................................................. 28 8.2.Guarantee of direct access to original documents ............................................................... 28 9.Quality Control and Quality Assurance matters .................................................................. 28 9.1.Monitoring ........................................................................................................................... 28 9.2.Audit .................................................................................................................................... 28 10.Ethical Considerations ........................................................................................................... 28 10.1.Rules and Regulations ...................................................................................................... 28 10.2.Handling of Personal Data ................................................................................................. 28 10.3.Provision of samples and information to other institutions ............................................... 29 10.4.Benefits, burdens and measures to minimize anticipated risks to subjects ............... 29 11.Handling and Storage of Records ......................................................................................... 29 11.1.Handling and storage of materials ..................................................................................... 29 11.2.Storage of Samples, etc. and Use of Samples, etc. by Other Institutions, ........................ 30 11.3.Data collection methods .................................................................................................... 30 12.Financial payments and compensation for the conduct of clinical research. ................... 30 12.1.Funding sources and financial relationships (conflicts of interest, COI) .......................... 30 12.2.Research-related costs ....................................................................................................... 30 12.3.Health damage compensation ............................................................................................ 30 13.Publication of information on clinical research. .................................................................. 31 14.Duration of Clinical Research ............................................................................................... 31 15.Matters relating to the explanation of and consent to study subjects ............................... 31 15.1.Preparation and revision of informed consent documents and consent forms .................. 31 15.2.Informed consent ............................................................................................................... 31 15.3.Informed assent.................................................................................................................. 32 15.4.In case of consent withdrawal ........................................................................................... 32 15.5.Reporting to the Clinical Research Review Committee, the Minister of Health, Labor and Welfare and the administrator of the implementing medical facility. ....................................... 33 15.5.1.Regular report ............................................................................................................. 33 15.5.2.Non-conformity Reporting ...................................................................................... 33 15.5.3.Report from the audit officer ................................................................................... 33 15.6.Changes to the study protocol ........................................................................................... 33 15.6.1.Amendments to Study Protocols ............................................................................. 33 15.6.2.Revision of Study Protocol ..................................................................................... 33 15.6.3.Subject explanation and re-consent for study protocol changes ................................. 33 15.7.Reffernses .......................................................................................................................... 34 15.8.Appendix ........................................................................................................................... 34 WISH-J study version 1.5 4/23/2025 3 mark Unabbreviated expressions eCRF Electronic Case Report Form electronic case report form EASI The Eczema Area and Severity Index EDC Electronic Data Capture FA Food allergy food allergy IgE Immunoglobulin E jRCT Japan registry of clinical trials Clinical Research Submission and Release System OFCs Oral Food Challenge Oral food load test POEM The Patient-Oriented Eczema Measure TARC Thymus and Activation-Regulated Chemokine Definition of a term test intervention Ingestion of the investigational food product (walnut powder). study intervention period From the start date of intake of the test food to the VISIT at 24 months of age (until the end date of intake) Weeks of study intervention period From the week of intake of the test food to the last week of the visit at 24 months of age. If the visit falls in the middle of the week, it must be the week prior to the Visit. test (testing) period From the date of registration to the date of the 24-month-old visit X months old Refers to the period from the date the subject reaches X months 0 days of age to the day before the (X + 1)-month birthday, based on the subject’s date of birth. legal representative Spouse, person exercising parental authority, guardian, or other similar person of the subject of this study Principal investigator Principal investigators representing more than one site participating in the study principal investigator A physician who oversees the work related to the study at each site participating in the study research physician Physicians who will share clinical research duties under the guidance of the principal investigator at each site participating in the study person who is being studied Persons who are considered to be the main subject of the study based on eligibility criteria and other factors. test subject Persons who have obtained consent, meet eligibility criteria and are participating in the study atopic dermatitis The U.K. Working Party’s diagnostic criteria for atopic dermatitis, developed by Williams et al. (Br J Dermatol 1994), require the presence of the major criterion and at least three minor criteria for diagnosis. immediate allergy Defined as the occurrence of any skin, respiratory, gastrointestinal, neurological, or circulatory symptoms included in the Sampson Severity Score (Table 2) within 120 minutes after ingestion of the suspected food, provided that other causes can be ruled out.. Maximum severity score The maximum severity score refers to the highest Grade (1–5) assigned using the Sampson Severity Score during the study WISH-J study version 1.5 4/23/2025 4 treatment period, as determined by the physician based on the subject’s diary and caregiver’s report. Anaphylaxis Anaphylaxis is defined as immediate allergic symptoms that are Grade 3 or higher on the Sampson severity score (Table 2). contact dermatitis Defined as skin symptoms around the mouth or in areas of direct contact within 15 minutes after ingestion of food. Food protein-induced enterocolitis syndrome Follows the diagnostic criteria of Novak et al. IgE sensitization An IgE sensitization is defined as a specific IgE antibody titer of 0.35 kUA/L or higher. POEM Score The Patient-Oriented Eczema Measure (POEM) consists of seven questions evaluating symptoms such as itching, dryness, redness, and sleep disturbance associated with atopic dermatitis. Parents or guardians complete the questionnaire weekly to assess disease severity. The POEM is recommended by the global Harmonising Outcome Measures for Eczema (HOME) initiative as a patientreported outcome measure. EASI Score The Atopic Dermatitis Severity Scale was developed by Hanifin et al. The EASI score is calculated by a physician blinded to the site, extent, and severity of atopic dermatitis lesions (0-72). The global Harmonising Outcome Measures for Eczema (HOME) initiative recommends the use of the EASI score as an outcome measure for atopic dermatitis in clinical trials. Table 1: The U.K. Working Party's diagnostic criteria (translated into Japanese by the Research Secretariat) Atopic dermatitis is diagnosed when the major criterion (1) and three or more minor criteria (2) are met. Main criterion An itchy skin condition (or parental report of scratching or rubbing in a child) Minor criteria History of involvement of the skin creases such as folds of elbows, behind the knees, front of ankles or around the neck (including cheeks in children under 10). A personal history of asthma or hay fever (or history of atopic disease in a first-degree relative in children under 4) A personal history of a general dry skin in the last 12 months. Visible flexural eczema (or eczema involving the cheeks/forehead and outer limbs in children under 4. Onset under the age of 2 (not used if child is under 4). Table 2: Sampson Classification Grade Skin Gastrointestinal Respiratory Cardiovascular Neurological 1 Itching, erythema, urticaria, vascular angioedema Oral discomfort, mild nausea Itchy throat, slight discomfort – – 2 Itching, erythema, urticaria, Vomiting, 1–2 episodes of vomiting/diarrhea Mild nasal congestion, runny nose, – Restlessnes s/anxiety WISH-J study version 1.5 4/23/2025 5 vascular angioedema , transient abdominal pain 1–2 sneezes, occasional cough 3 Above symptoms Repeated vomiting/diarrhea , persistent abdominal pain Persistent nasal congestion, repeated sneezing, continuous coughing, wheezing Tachycardia (HR ≥15 bpm above normal) Anxiety 4 Above symptoms Above symptoms Laryngeal stridor, barking cough, severe dyspnea, cyanosis, respiratory distress Arrhythmia, hypotension Agitation, sense of impending doom 5 Above symptoms Above symptoms Respiratory arrest Severe hypotension, cardiac arrest Loss of consciousness WISH-J study version 1.5 4/23/2025 6 1．Key contacts. 1.1. Principal investigator Kiwako Yamamoto, Doctor, Department of General Allergy, Allergy Center, National Center for Child Health and Development 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan Tel: 03-3416-0181 (ext. 7910) E-mail: [email protected] 1.2. Participating institutions and principal investigators Kiwako Yamamoto, Doctor, Department of General Allergy, Allergy Center, National Center for Child Health and Development Tsutomu Natsume, Assistant Professor, Department of Pediatrics, Hamamatsu University Hospital Fumiya Yamade, Associate Professor, Department of Pediatrics, Narita Hospital, International University of Health and Welfare Chikako Motomura, Medical Director, Department of Pediatrics, National Hospital Organization Fukuoka Hospital Sayaka Hamaguchi, Doctor, Department of Pediatrics, Tokyo Metropolitan Hiroo Hospital 1.3. Research collaborator See list of participating researchers 1.4. Research administration, coordination and management staff Department of General Allergy, Allergy Center, National Center for Child Health and Development, Mayako Saito 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan Tel: 03-3416-0611 (ext. 7910) E-mail: allergy_res[email protected] 1.5. Statistician Medical Support Center for the Japan Environment and Children's Study, National Center for Child Health and Development, Limin Yang (ext. 7877). 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan Tel: 03-3416-0181 1.6. Assignment and allocation Medical Support Center for the Japan Environment and Children's Study, National Center for Child Health and Development, Limin Yang (ext. 7877) 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan Tel: 03-3416-0181 1.7. Data management Allergy Center, National Center for Child Health and Development, Kumiko Watanabe 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan Tel: 03-3416-0181 (ext. 7661) 1.8. Monitoring Head of Unit, Monitoring Unit, Data Science Division, Hospital Clinical Research Center, National Center for Child Health and Development, Mayumi Sako 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535, Japan Tel: 03-3416-0181 (ext. 7551) 1.9. Chief auditor N/A 1.10. Walnut powder provider B-Case Inc. Representative director Tetsuaki Kon 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan Tel: 03-5665-2311 WISH-J study version 1.5 4/23/2025 7 2. Background and rationale. 2.1. Background 2.1.1 Prevention of food allergies through early intake Food allergy is increasing worldwide, and once developed, requires dietary restrictions until remission, which has a significant impact on the lives of the individual and their caregivers. In recent years, the dual-allergen-exposure hypothesis has been proposed as the mechanism of food allergy development.1) This hypothesis suggests that in patients with atopic dermatitis, early exposure to allergens through the gut (via eating) promotes immune tolerance, while initial exposure through the skin leads to allergic sensitization. Based on this hypothesis, it has been suggested that preventing percutaneous sensitization by maintaining healthy skin during infancy and inducing immune tolerance through ingestion of food antigen proteins from an early age may have a preventive effect against food allergy development. In fact, as an oral intervention, recent data from randomized controlled studies have shown that the onset of allergy can be prevented by early initiation of oral intake of foods in infancy, such as hen eggs2), peanuts3), and milk4). Interventions in which multiple foods are consumed simultaneously have also been reported 6) (Table 1). Table 1: Randomized controlled trials of food allergy prevention by early intake food targe t grou p Presen ce or absenc e of eczem a in the subjec t Interven tion details (Protein content) Interven tion period outco me Prevalence of both groups Placebo group vs. intervention group Country/Regio n/Year ITT PPS hens egg High -risk infan t (Fam ily histo ry) ± (Proact ive treatm ent) Heated whole egg powder (25 mg → 125 mg/day) From 6 months to 12 months of age Chick en egg allerg y at 12 month s of age 38% vs. 8%. (p=0.00 01) 38% vs 4% (p<0.00 01) 2017, Japan2) pean uts Infan ts with sever e AD, EA or both ± Snacks with peanuts or butter (6g/week ) From 410 months old to 60 months old Peanu t allerg y at 60 month s of age 17.2% vs. 3.2 (p<0.00 1) 17.3% vs. 0.3 (p<0.00 1) .2015, United Kingdom3) (cow' s) milk Gene ral infan t ± (Proact ive treatm ent) Milk powder >= 10ml/day (150 mg/day) From 1 month to 3 months of age Cow's milk allerg y at 6 month 6.8% vs. 0.8 (p<0.00 1) 8.7% vs. 0.0 (p<0.00 1) .2021, Japan 4) WISH-J study version 1.5 4/23/2025 14 introduction during infancy have already been demonstrated in randomized controlled trials, and prevention effects have been reported in multi-allergen intervention studies⁵⁾⁶⁾ (although walnuts were not included), suggesting that early introduction can be expected to have allergy prevention effects for any food. American dietary guidelines⁸⁾ recommend nut consumption during weaning. In our clinical practice, we also recommend early introduction of nuts in powder or paste form. Therefore, setting a placebo group without consumption would not align with current practices. However, since nuts including walnuts do not have a tradition of early introduction during infancy, far fewer families can actually start early introduction in clinical practice compared to hen's eggs (Nutrients, 2024, accepted). Additionally, walnuts tend to be sensitized later than hen's eggs (Clin Exp Allergy, 2024, accepted). Considering these circumstances, we set up a non-inferiority trial comparing sensitization suppression effects by introduction timing, randomizing subjects to groups before or after weaning completion. Group B (walnut introduction after weaning completion) in this trial will completely avoid walnuts until a maximum of 14 months of age, but considering Japan's infant walnut consumption patterns and the suggested later onset of sensitization, this is considered within an acceptable range. Criteria for subject selection, exclusion, and study discontinuation Subjects meeting the inclusion criteria and do not conflict with all of the exclusion criteria will be enrolled in this study. 3.4. Subject inclusion criteria. 1. Children aged 5-7 months at the time of consent. 2. Children diagnosed, or judged to have developed atopic dermatitis before 6 months of age using the UK Working Party diagnostic criteria at the time of consent (including children who have already started treatment and are in remission). 3. Children whose legal representatives have provided written informed consent after receiving explanation and achieving sufficient understanding of study participation. [Rationale] 1), 2) Because this study aims to demonstrate non-inferiority of the efficacy of intervention from early or complete weaning for infants at high risk of developing food allergies. 3.5. Exclusion criteria 1. Children born at less than 37 weeks of gestational age. 2. Children with previous experience consuming walnuts or walnut products. 3. Children with suspected or pre-existing food protein-induced gastroenteropathy 4. Children suffering from illnesses that may interfere with the study (e.g. an underlying illness that requires tube feeding or an underlying illness that is likely to result in prolonged hospitalization during the study period for treatment or surgery). 5. Children whose legal representatives are unable to communicate adequately in Japanese 6. Children deemed ineligible by the principal investigator, sub-principal investigator or co-investigator. [Rationale] 1)-4) Due to concerns about the impact on efficacy evaluation; 4)-6) Due to concerns about the impact on safety or ethical considerations. 3.6. Study intervention discontinuation and participant discontinuation/withdrawal. The principal investigator or co-investigator will discontinue the study food intake if he/she decides that the study cannot be continued for any of the following reasons. The date and reason for discontinuation will be recorded in medical records and electronic case report form (eCRF), and any necessary tests will be conducted at discontinuation to assess efficacy and safety (if discontinuation occurs prior to study intervention, efficacy and safety will not be assessed). If discontinuation is due to illness, follow-up will continue as far as possible until the subject’s recovery. 1) Inability to start the study intervention within the specified period for the assigned group. WISH-J study version 1.5 4/23/2025 15 2) Positive result in OFC of the test food conducted during the study intervention period. 3) Difficulty continuing study intervention due to adverse events during study intervention period. 4) Withdrawal of consent by the subject's legal representative. 5) Discovery after registration that inclusion criteria are not met or that exclusion criteria are violated. 6) Difficulty continuing the study due to illness or other medical condition. 7) Subject does not visit due to relocation or other reasons. 8) Other situations where study discontinuation is deemed appropriate by the principal investigator, sub-principal investigator or co-investigator. 9) 3.7. Study discontinuation criteria. The principal investigator will consider whether or not to continue the study if any of the following apply. If the efficacy and safety evaluation committee or the certified clinical research review committee recommends or instructs discontinuation, the study will be discontinued. Within 10 days of deciding early study termination, the principal investigator will submit a discontinuation notice to the certified clinical research review committee and submit a specified clinical research discontinuation report to the Minister of Health, Labor and Welfare. The administrator of the implementing medical institution will be promptly notified in document with the reason for the discontinuation, and subjects' legal representatives will be promptly notified with appropriate post-processing guaranteed. 1) Obtaining significant information on the quality, safety or efficacy of the test food. 2) Difficulty recruiting research subjects or high dropout rates making study completion difficult. 3) Instructions for study protocol changes from the efficacy and safety evaluation committee or certified clinical research review committee that are deemed difficult to accept. 4. Matters relating to the treatment of clinical research subjects. 4.1. Subject registration procedures This study uses an Electronic Data Capture system (EDC) for subject registration and randomized allocation (NCCHD REDCap system). 1) The principal investigator or co-investigator obtain written consent from legal representatives (parents) using designated forms and confirm all inclusion criteria are met and no exclusion criteria are violated. 2) The principal investigator or co-investigator enter subject registration information (yes/no for each inclusion criterion, yes/no for each exclusion criterion) into EDC for eligible candidates. A subject identification number is assigned and an allocation to one of two groups occurs. 3) Once registered, the NCCHD REDCap system sends a email confirmation notifications to the research office, principal investigator, sub-principal investigator, co-investigators and the monitoring personnel. 4) The principal investigator or co-investigator confirms the allocation results and informs the subjects of visit 2 (start of the study intervention) timing. [Registration notes]. 1) Ineligible subjects and those who withdraw consent before registration are not entered into NCCHD REDCap. 2) Registration is complete upon receiving the confirmation email notification. 3) Promptly contact the research office for erroneous registration, duplicate registration, consent withdrawal, discontinuation or dropout. 4) For duplicate registrations, the first registration takes priority. WISH-J study version 1.5 4/23/2025 16 4.2. Allocation method and adjustment factors Subjects are allocated 1:1 to the early weaning walnut initiation group (group A) and the weaning completion walnut initiation group (group B) using permutated block randomization in registration order. No adjustment factors are used. 4.3. Blinding. The study interventions in this study will not be blinded to the subjects, principal investigator, sub-principal investigator and co-investigators. 4.4. Emergency unblinding. Not applicable to this study. 4.5. Study intervention The study intervention in this study is the consumption of test food. The principal investigator or co-investigator instructs the legal representatives (parents) or caregivers on how to consume the test food and how to record the diaries at the time of registration. The test food will be distributed to the legal representatives or caregivers and OFC will be conducted at each facility’s outpatient clinic for safety reasons at the start of intake (visit 2) and at the time of increased intake (visit 3, visit 4). In both groups, an OFC assessment of daily walnut intake (4 g in total) will also be conducted at visit 6 for those who wish. Details of these OFCs will follow a separate instruction manual. Investigators check diary consumption records at each visit. When test food is given to children, consumption is considered to have occurred if more than one mouthful is swallowed (explain to legal representatives to mix with amounts the subject can finish eating). If vomiting or spitting up occurs after test food consumption, re-administration on the same day is not performed regardless of time from consumption to vomiting (follow the response flow for allergic symptom onset after investigational food consumption [Appendix 1]). However, during initial or dose escalation OFC at outpatient clinics, re-administration may occur at investigator’s discretion. Study intervention periods are as follows. For early weaning walnut initiation group (Group A): study intervention starts between 6-8 months of age until visit 6 at 24 months or discontinuation date. For weaning completion walnut initiation group (Group B): study intervention starts between 12-14 months of age until visit 6 at 24 months or discontinuation date. For those who wish to undergo daily walnut consumption OFC at visit 6, study intervention ends upon OFC completion for both groups. [Walnut intake capacity, method and duration] ① Early weaning walnut initiation group (group A) <Visit dates for walnut powder initiation and dose escalation> Visit 2: When subject is 6-8 months old and within 6 weeks of visit 1 Visit 3: Visit 2 date + 8 weeks (± 2 weeks) Visit 4: Visit 2 date + 16 weeks (±2 weeks) ⚫ Step 1: From the start of visit 2 intervention until the day before visit 3, 1 packet (0.8 g) of walnut powder (containing 2.5 mg of walnut protein)/dose ⚫ Step 2: From the day of visit 3 to the day before visit 4, 1 packet (0.8 g) of walnut powder (containing 7.5 mg of walnut protein) /dose ⚫ Step 3: From the day of visit 4 to the day of visit 6, 1 packet (2.2 g) of walnut powder (containing 20 mg of walnut protein)/dose Consume once a day, 3-7 days per week. [Rationale]. Regarding the safety and feasibility of the amount of walnut protein in test food, there is a pilot study using mixed powder of 10 allergens including walnuts (30mg, 90mg, 300mg of each food protein) for early introduction (n=45)10)11). All subjects were able to consume safely with no serious adverse events after 2-12 months of introduction. Therefore, we considered up to a maximum 300 mg protein dose to be safely consumable. For food allergy prevention effects, WISH-J study version 1.5 4/23/2025 17 while there are no walnut intervention studies, we referred to hen's egg allergy prevention studies2) (protein amount 25mg→125mg in 2 stages) and other multi-allergen intervention studies6) (hen's egg protein 2.2mg→6.6mg→17.6mg, peanut protein 0.6mg→1.8mg→4.8mg), and evidence from clinical practice showing minimal oral consumption can be effective for immune induction19). The amount of walnut proteins were set at 2.5mg, 7.5mg, 20mg in 3 stages based on allergy expert opinions and manufacturing capabilities of nut powder companies, as well as taking into consideration the safety and minimal effective amounts (equivalent to approximately 0.017g, 0.051g, 0.137g walnut respectively, assuming 146mg protein per 1g walnut). The frequency of 3 or more times per week was set considering that peanut prevention intervention studies³⁾ and 6-allergen prevention intervention studies⁵⁾ showed sufficient prevention effects with 2-3 times per week consumption when adherence was good, while also considering caregiver burden for interventions lasting up to 1.5 years. [Common items for both groups] ・Consumption method: The test food is taken once a day. The intake should be mixed with other foods such as porridge. To prevent consumption residue, explain to legal representatives to mix with amounts the subject can finish eating. ・Subjects must not consume walnuts or products containing walnuts, other than the test foods and the OFC in this study, from the time of obtaining study consent until the end or discontinuation of the study intervention. However, for breastfeeding mothers, there is no need to avoid remove walnuts or products containing walnuts. ・Acceptable consumption frequency: At least 3 days per week. ・Interruption of consumption during illness: Test food should not be consumed during illnesses such as gastroenteritis, fever, etc. Interruption of consumption of the test food due to temporary illness with the expectation of resuming normal consumption does not fall under criterion 3 for discontinuation of the study. After symptom improvement, consumption should be resumed at the same amount of walnut powder that was consumed before the temporary interruption. ・Test foods are distributed in outpatient clinics and delivered by the respective medical institution as appropriate for cases where it is difficult or unnecessary to see the patient in the step 3 period. ・In case of allergic symptoms due to consumption of test food. If allergic symptoms are suspected due to consumption of test food, legal representatives or caregivers should temporarily stop consumption, follow the response flow for allergic symptom onset(Appendix 1), and contact the consultation service at the respective medical facility. The principal investigator or co-investigator receiving the contact will determine if emergency visits are necessary. However, when contact dermatitis suspected symptoms appear after consumption of the test food, the test food should be applied to the lips and around the mouth before consumption for prevention, and retry consumption again the next day. If symptoms do not appear, continue consumption. If symptoms persist, temporarily stop consumption and contact the consultation service at the respective medical facility, as in the case of other symptoms. ・Antihistamines and white petrolatum will be prescribed at the time of registration and, if necessary, will be used in accordance with the flow in the event of allergic symptoms (Appendix 1), or under the instruction of the principal investigator, co-investigator. ・Treatment of atopic dermatitis will be provided within the scope of insurance coverage for routine care at each facility. 4.6. Observations/test items and treatment information to be reported The principal investigator, co-investigator will conduct observations, tests and evaluations according to the time schedule and collect data in an electronic case report form (eCRF). If the study intervention is discontinued, data will be collected at that point for the end-of-study (at 24 months of age) endpoints. If the intervention is discontinued due to illness, follow-up continues as far as possible until the subject recovers to its normal status. WISH-J study version 1.5 4/23/2025 18 <Schedule Table>. duration of the intervention (e.g. in a clinical trial) At the end or discontin uation of the study intervent ion Visit 1 2 3 4 5 temporar y medical examinat ion 6 Setting period (Group A) 5-7 months old At 6-8 months of age and within 6 weeks of visit 1 +8 +/- 2 weeks after Visit2 +16 +/- 2 weeks after Visit 2 At 1214 months of age At 24 months of age (±4 weeks) or date of discontin uation Setting period (Group B) At 12-14 months of age +8 +/- 2 weeks after Visit2 +16 +/- 2 weeks after Visit 2 +24 +/- 2 weeks after Visit 2 At 24 months of age (±4 weeks) or date of discontin uation Obtainin g Consent ○ Registrati on and allocatio n 1) ○ Age of onset of atopic dermatiti s 2) ○ Backgrou nd and living environm ent of subjects and blood ○ WISH-J study version 1.5 4/23/2025 19 relatives 3 Walnut intake in the surroundi ng area 4) ○ ○ (Group B only) ○ (Group A only) ○ Nutrition al intake status 5) ○ ○ Complica tions/Hist ory ○ ○ OFC at the impleme nting medical facility6) ○ Test food start OFC ○ Test food bulking OFC ○ test food Increase d OFC ○ (Tempor ary OFC) ○ (daily intake of walnuts OFC: on request) Ingestion of test food Step 1 Step 2 Step 3 Step 3 Step 3 Height and Weight ○ ○ ○ ○ ○ ○ EASI Score(7) ()) ○ ○ ○ ○ ○ ○ POEM score(8) ()) ○ ○ ○ ○ ○ ○ Caregive r Burden Level on Test Interventi on (9) ()) ○ ○ ○ ○ History of immediat e walnut allergy (10) ()) ○ ○ ○ ○ ○ ○ History of food proteininduced gastroent eropathy (1) (1) ()) ○ ○ ○ ○ ○ ○ WISH-J study version 1.5 4/23/2025 20 Presence of expirator y wheezing (1) (2) ()) ○ Allergic rhinitis (1) (3) () ○ Presence of immediat e food allergies other than walnuts (1) (4) ()) ○ Blood test (1) (5) ()) ○ (Group B only) ○ (Group A only) 0 ○ Check logbooks (1) (6) ()) ○ ○ ○ ○ adverse event ○ ○ ○ ○ ○ ○ <Detail of survey items>. Collected information is entered into eCRF as data for the study. 1) Registration/allocation: Date of consent, date of birth and eligibility information registered in NCCHD REDCap system for allocation. 2) Atopic dermatitis onset age: Age at diagnosis based on UKWP criteria, collected through medical history-taking. 3) Subject and family background: From medical records and questionnaires. Subject background: gender, age (months) at registration →From medical records. Gestational weeks, birth height, birth weight, mode of delivery (vaginal delivery/cesarean section), race → From questionnaires. Subject's maternal background: History of allergic diseases → From questionnaires. Subject's paternal background: History of allergic diseases → From questionnaires. Background of subjects' siblings: number of siblings, age, sex of siblings, history of allergic diseases → From questionnaires Pets (dogs, cats, other) within the residence, smoking by family members → From questionnaires 4) Surrounding walnut consumption status: From questionnaires ・Frequency and quantity of walnut intake of family members living together ・Household situation of paternal and maternal parents ・Frequency of visits ・Frequency and quantity of walnut intake 5) Nutritional intake status: From nutritional record forms. ・Intake of breast milk, general milk, allergy milk WISH-J study version 1.5 4/23/2025 21 ・Weaning or not and start date ・Intake of following foods (and if so, its’ starting date) walnut foods (other than the test food)/eggs/milk (dairy products other than milk powder)/wheat/soy/peanut/cash/ hazel/almond products 6) OFC at the participating institute: (1) Test food initiation OFC (visit 2) and dose escalation OFC (visits 3 and 4): 1 packet of walnut powder (containing 2.5 mg of walnut protein) in visit 2, 1 packet of walnut powder (containing 7.5 mg of walnut protein) in visit 3 and 1 packet of walnut powder (containing 20 mg of walnut protein) in visit 4, each in the form of a single loading dose. (2) Emergency OFC: When allergic symptoms are suspected during home consumption and principal investigator or co-investigator decides that it's necessary on emergency visit, singledose challenge will be conducted with 1 packet of walnut powder (dose at the step level when symptoms occurred). (3) Daily walnut intake OFC (visit 6): For volunteers at visit 6, 40-minute divided-dose OFC with walnut equivalent to 1 walnut (4g) (dose divided into 1g-3g) For OFC(1)-(3), the decision is made according to the PRACTALL criteria given in Table 3. 20) and is considered positive if any of the following criteria (1) to (3) are met within 120 minutes of the last intake of the food. ➀ One [A] symptom category shown in Table 3 ② Three [B] symptom categories shown in Table 3 ③ One [B] symptom category shown in Table 3 persists The principal investigator or co-investigator collects from the electronic medical record the date of OFC, time of intake, highest score for each category by site, overall decision time, test result, basis for decision, and persistent symptoms if one [B] symptom category persists. 7) EASI (Eczema Area and Severity Index) score: The principal investigator or co-investigator determines the location, extent and severity of atopic dermatitis, calculates the EASI score and collects each assessment score from the electronic medical record. 8) POEM (Patient Oriented Eczema Measure) score: POEM consists of seven questions assessing skin dryness, redness sleep disturbance, etc. due to atopic dermatitis. Information is collected from questionnaires answered by legal representatives or caregivers. 9) Level of burden on caregivers with regard to the study intervention: The principal investigator or co-investigator conduct multiple-choice questioning(very much/quite a lot/slightly/not at all) at each visit to collect information. 10) Immediate-type walnut allergy history: Principal investigator/co-investigators collect presence/absence of symptoms and maximum severity scores using Sampson classification during the period from previous visit to current visit through history-taking, diary confirmation, and emergency visits for investigational food consumption when necessary. 11) History of food protein-induced enterocolitis syndrome: Principal investigator/coinvestigators collect through history-taking whether Acute FPIES, Chronic FPIES, FPE, FPIAP, or "other" not fitting any category occurred during the period from previous visit to current visit, with "yes/no" responses, and for "yes" cases: "diagnosis/suspected," presence/absence of each diagnostic definition item, and suspected foods (multiple selections possible) from electronic medical records (see Appendix 2 for definitions). 12) Presence or absence of expiratory wheezing: Principal investigator/co-investigators collect through history-taking the presence/absence of occurrence during the study intervention period. 13) Presence or absence of allergic rhinitis: Principal investigator/co-investigators will collect information through history-taking the presence/absence of the condition diagnosed during the study intervention period. WISH-J study version 1.5 4/23/2025 22 14) Presence or absence of immediate-type food allergy other than walnuts: Principal investigator/co-investigators collect through history-taking the presence/absence of disease names and causative foods. 15) Blood tests: Collect 3ml whole blood to measure total IgE antibody levels, specific IgE antibody levels (walnut, Jug r 1), and serum TARC values. Total IgE and specific IgE antibody levels are measured by ImmunoCAP method. Principal investigator/coinvestigators collect results from electronic medical records. 16) Diary records 4 weeks per page, with legal representatives or caregivers recording: presence/absence of allergic symptoms (perioral symptoms, systemic symptoms), other food consumption status during symptomatic periods, test food consumption status (presence/absence of consumption, reasons for inability to consume 3+ times per week [multiple choice: ①forgot consumption, ②child's refusal, ③other (free text)]). For systemic symptoms, complete systemic symptom onset records (see Appendix: Diary). Principal investigator/co-investigators collect completed diary pages (4-week intervals) at relevant visits and record: presence/absence of diary confirmation, diary confirmation date, collection period (weeks) for recovered diary pages, total weeks achieving 3+ times per week consumption within collection period. Table 3 PRACTALL criteria I. Skin Skin Skin A. Erythematous rash: % area involved (see body surface area diagram) Erythematous rash: % area involved (see body surface area diagram) 0 = Absent / None 1 = Mild few areas of faint erythema / 1-3 locations, 5-10% as range 2 = Moderate areas of erythema / 10%<, ≤50 3 = Severe generalized marked erythema / >50 Figure Law of 5 B.Pruritus pruritus 0= Absent / None 1= Mild, occasional scratching 2= Moderate: scratching continuously for >2 minutes at a time 3= Severe: hard continuous scratching - excoriations / Severe: hard continuous scratching, epidermal excoriations [B]. C. Urticaria / angioedema Urticaria: Angioedema 0= Absent / None 1= Mild: less than 3 hives, or mild lip edema / Mild: less than 3 hives, or mild lip swelling [B] 2= Moderate: more than 3 and less than 10 hives, or significant lip or face edema / Moderate: more than 3 and less than 10 hives, or significant lip or face swelling [A]. 3= Severe: generalized involvement / Severe: generalized [A]. WISH-J study version 1.5 4/23/2025 23 D. Rash rash 0= Absent / None 1= Mild: few areas of faint erythema / Mild: small areas of faint erythema 2= Moderate: areas of erythema/ Moderate: erythema present [B]. 3= Severe: generalized marked erythema (>50%)/ Severe: erythema more extensive than 50% [A]. II. Upper respira tory Upper respira tory tract A. Sneezing/ Itching Sneezing, itching 0= Absent / None 1= Mild: rare bursts, occasional shuffling/ Mild: rare sneezing, occasional sniffling 2= Moderate: bursts<10, intermittent rubbing of nose ,and/or eyes or frequent sniffing/ Moderate: sneezing less than 10 times, intermittent rubbing of nose or eyes, frequent sniffing [B] 3=Severe: continuous rubbing of nose and/or eyes, periocular swelling and/or long bursts of sneezing, persistent rhinorrhea Severe: continuous rubbing of nose and/or eyes, periocular swelling and/or long bursts of sneezing, persistent rhinorrhea [A]. III. Lower respira tory Lower respira tory tract A. Wheezing Wheezing 0= Absent / None 1= Mild: expiratory wheezing to auscultation / Mild: expiratory wheezing on auscultation [A]. 2= Moderate: inspiratory and expiratory wheezing [A]. 3= Severe: use of accessory muscles, audible wheezing/ Severe: use of respiratory accessory muscles, wheezing audible without stethoscope [A]. B. Laryngeal larynx 0= Absent / None 1= Mild: >3 discrete episodes of throat clearing or cough, or persistent throat tightness/pain/ Mild: series of 4 or more cough episodes or persistent throat tightness/pain [B]. 2= Moderate: hoarseness, frequent dry cough / Moderate: hoarseness, frequent dry cough [A]. 3= Severe: stridor / Severe: inspiratory wheezing [A]. IV. Gastroi ntestin al Gastroi ntestin al A. Subjective Complaints Subjective Complaints 0= Absent / None 1= Mild: complaints of nausea or abdominal pain, itchy mouth/throat/ Mild: nausea, abdominal pain, itchy mouth or throat [B] 2= Moderate: frequent complaints of nausea or pain with normal activity/ Moderate: frequent nausea or abdominal pain (no decrease in activity) [B]. 3= Severe: notably distressed due to GI symptoms with decreased activity/ Severe: suffering significantly from GI symptoms (with decreased activity) [B]. B. Objective Complaints Objective Symptoms 0= Absent / None 1= Mild: 1 episode of emesis or diarrhea / Mild: 1 episode of vomiting or diarrhea [B] 2= Moderate: 2-3 episodes of emesis or diarrhea or 1 of each / Moderate: 2-3 episodes of emesis or diarrhea or 1 of each [A] WISH-J study version 1.5 4/23/2025 30 6) Documents containing an overview of medicines and other products used in specified clinical trial and records relating to medicines and other products. 7) Other documents necessary to carry out specified clinical trial. 11.2. Storage of samples, etc. and use of samples, etc. by other institutions. The principal investigator will properly store the subject's samples and other materials obtained in the study for a period of at least five years from the date of study completion. Residual blood (serum) from specimens examined at NCCHD testing centers will be frozen and stored at the Allergy Center sample storage center. All specimens from subjects at other institutions are disposed by appropriate means. If residual samples are to be used for future research that is not identified at the time consent is received, separate research subject explanation and consent are obtained before use. 11.3. Data collection methods Study-related data is collected via Electronic Case Report Forms (eCRF) using the NCCHD REDCap system in formats not including personally identifiable information. Responsible physicians and co-investigators at each collaborating medical facility enter data into eCRF after each visit completion. Subjects' legal representatives record in diaries. 12. Financial payments and compensation for the conduct of clinical research. 12.1. Funding sources and financial relationships (conflicts of interest, COI). The study will be funded by the Research and Development Fund for Growth and Development (2023B-14) and will also be conducted as a collaborative study using test foods provided by B-Case Co., Ltd. B-Case Co., Ltd. will be involved in part of the conduct of the study (provision of test food and delivery to subjects), but not in the analysis of the study results. The state of COI due to joint research will not affect the planning and conduct of the study, the results and interpretation of the study, and the conduct of the study will not harm the rights and interests of the study subjects. Regarding the management of COI with the company concerned, the principal investigator has submitted a COI management standard and management plan to certified clinical research review committee for approval, in accordance with the guidance on COI management for clinical research in the “Clinical Research Act”. Each researcher appropriately manages COI related to this study in accordance with the COI management standards and management plan, and disclose them appropriately upon request of the academic societies and medical journals where the study results are scheduled to be published. 12.2. Research-related costs The test food in the study was provided by B-Case Co., Ltd., and the tests (blood tests for IgE antibodies, etc.) and drug prescriptions are conducted within normal insurance practice coverage, paid through subject's health insurance or self-payment. To reduce burdens associated with clinical research participation, Quo cards worth 2,000 yen per visit are provided to subjects as participation cooperation fees. 12.3. Health damage compensation When health damage occurs due to this trial, treatment is provided using subjects' health insurance with best efforts as in normal medical care. For this trial, clinical research insurance is purchased with the principal investigator, co-investigators, and all personnel involved in this trial as insured parties to prepare for compensation for health damage occurring to subjects. This insurance pays insurance money for damages incurred when insured parties bear compensation responsibility or legal liability when health damage (death or grade 1 or 2 sequelae according to drug adverse reaction damage relief system standards) occurs to subjects due to clinical research. 13. Publication of information on clinical research. The study will be registered in the database (jRCT: Japan Registry of Clinical Trials) maintained by the Ministry of Health, Labor and Welfare (MHLW) prior to the start of the study. Study results belong to the NCCHD. The main study results will be submitted to academic journals after the final analysis is completed. In principle, the first author of the main article of the study results will be determined by the research office. Co-authors will be determined by the WISH-J study version 1.5 4/23/2025 31 principal investigator in accordance with the Uniform Requirements for Manuscripts Submitted to Biomedical Journals of the International Committee of Medical Journal Editors. All coauthors should review the content of the article prior to submission and agree on the content of the publication. Any researcher who disagrees with the content will be discussed and, if agreement is still not reached, the study office may choose not to include that researcher as a coauthor. 14. Duration of clinical research. Subject registration period: From the date of publication of the clinical study in the jRCT until 30 September 2026. Total study period: From the date of publication of the clinical study in the jRCT to 30 November 2029. If the implementation period is extended for reasons such as not reaching the target number of cases within the research implementation period, the extension is subject to approval by certified clinical research review committee. 15. Matters relating to the explanation of and consent to study subjects. 15.1. Preparation and revision of informed consent documents and consent forms Informed consent documents and consent forms are prepared by the principal investigator and used after approval by certified clinical research review committee. If revisions are made, they are reapplied to the certified research review committee and used after approval is obtained. The principal investigator will promptly revise the informed consent documents when information is obtained that may influence the subjects’ legal representatives’ intention to continue participating in the research. 15.2. Informed consent The study will be conducted after review and approval by the certified clinical research review committee and publication in the jRCT. Prior to the commencement of the study, the principal investigator and co-investigators provide clear explanations to candidate legal representatives based on informed consent documents including all items below according to the Clinical Research Act, obtaining written consent for trial participation based on legal representatives' free will after achieving sufficient understanding. When obtaining consent, sufficient time and opportunity for questions are provided to legal representatives' to enable them to decide whether or not to participate in the study, along with sufficient answers to questions. If consent is obtained, the subjects' legal representatives' and the principal investigator or co-investigator should sign and date the consent form. A copy of the consent form and the informed consent documents should be given to the patient (subject), while the original consent form should be kept at the collaborating medical facilities. Explanation includes that consent can be withdrawn at any time even after initially given, and that no disadvantages result from consent withdrawal. Principal investigators and co-investigators promptly explain to legal representatives and confirm study continuation intentions when new important information regarding walnut powder safety is obtained or when important information that may affect subjects' legal representatives' consent intentions is obtained. Principal investigators or co-investigators record explanation dates, explainers, explanation content, continuation intentions, and confirmation dates in medical records. [Explanatory Items] ① Study name, approval by collaborating medical institution and study plan has been submitted to the Minister of Health, Labor and Welfare. ② Name of the implementing medical institution and name and title of the principal investigator. ③ Reasons for selection as a subject for this study. ④ Benefits and disadvantages of the study. ⑤ Study participation refusal is voluntary. ⑥ Withdrawal of consent. WISH-J study version 1.5 4/23/2025 32 ⑦ No treatment disadvantage by participation refusal or consent withdrawal. ⑧ Study information publication methods. ⑨ Availability of research protocols and other research implementation materials upon request by research subjects or legal representatives and acquisition or inspection methods ⑩ Protection of personal data of subjects of this study. ⑪ Specimen storage and disposal methods. ⑫ Conflicts of interest including funding from pharmaceutical manufacturers and other compensation. ⑬ System for responding to complaints and enquiries. ⑭ Costs related to the conduct of this research. ⑮ Availability and content of other treatments and comparison with the expected benefits and disadvantages of other treatments. ⑯ Compensation and provision of medical care for health damage caused by the conduct of this study. ⑰ Review matters at the certified clinical research review committee that performs review opinion duties for this study and other matters relating to the certified clinical research review committee for this study. 15.3. Informed assent Since trial subjects are infants, informed assent is not conducted. 15.4. In case of consent withdrawal. If the subjects' legal representatives request consent withdrawal, this will be handled in accordance with the following. The study investigator at each site, reports to the principal investigator, and confirms the withdrawal of consent to participate in the study by the subject's legal representative using a withdrawal of consent form. Consent withdrawal includes participation consent withdrawal (withdrawing participation only while not deleting data up to withdrawal for analysis use) and complete consent withdrawal (withdrawing research participation and data use). Either withdrawal type results in deletion of personal information (names, addresses) stored at each implementing medical facility at withdrawal. In the event of withdrawal of consent to participate, the principal investigator (or co-investigator) at each site asks the research office to withdraw consent to participate in the study and dispose any remaining samples. The research office will request the sample storage center to dispose residual blood (serum) in the sample storage center. In the case of complete withdrawal of consent, the principal investigator (or co-investigator) at each site will ask the research office to withdraw consent to participate in the study and dispose any relevant data. The research office asks the sample storage center to dispose the relevant subject's data and samples, the data management officer excludes the withdrawn subject's data from the analysis data set and the sample storage center dispose residual blood (serum). 15.5. Reporting to the Clinical Research Review Committee, the Minister of Health, Labor and Welfare and the administrator of the implementing medical facility. 15.5.1. Regular report Principal and co-investigators conduct regular reports to clinical research review committees within 2 months after each 1-year period completion from implementation plan submission to the Minister of Health, Labour and Welfare (regional bureau), after reporting to implementing medical institution managers. After reporting to the Clinical Research Review Committee, the principal investigator provides information to each principal investigator, who reports the content of the information provided by the principal investigator to the administrator of the implementing medical institution. Additionally, principal investigators submit regular reports to the Minister of Health, Labour and Welfare (regional bureau) within 1 month of receiving clinical research review committee opinions. WISH-J study version 1.5 4/23/2025 33 15.5.2. Non-conformity Reporting When research non-compliance with regulations, study protocols, or procedures, or research data falsification or fabrication becomes apparent, co-investigators who become aware report to responsible physicians, who report to administrators of implementing medical institutions and principal investigators. Principal investigators provide information to all responsible physicians. When principal investigators determine serious non-compliance, they promptly seek clinical research review committee opinions. Among non-compliance, instances of not following study protocols due to urgent danger avoidance for subjects or other medically unavoidable reasons are not included in serious noncompliance. The following cases are determined as serious non-compliance: • Serious eligibility/exclusion criteria violations • Discontinuation criteria violations that may threaten subject safety • Data fabrication or falsification • Subject registration without consent (including consent form loss) • Other instances deemed serious by principal investigators 15.5.3. Report from the audit officer. No audits are planned. 15.6. Changes to the study protocol When the content of the study protocol is changed, the principal investigator reports to the administrator of the implementing medical facility and submits a notification of changes (Form 2), the revised implementation plan (jRCT) and the notification of the approved clinical research review committee to the Minister of Health, Labor and Welfare. The principal investigator provides information on any changes to the study protocol to the principal investigators at each site. The principal investigator reports the content of the information provided to the administrator of the site. 15.6.1. Amendments to the study protocol 1) May increase the risk to patients participating in the study. 2) Substantial impact on the study's primary outcome. 3) Essential impact on the system for conducting the study. A partial change to a study protocol that corresponds to one or more of the above is defined as an amendment. Version number upgrades of the study protocol and the informed consent documents due to revisions are indicated at the rank of 1, such as 2.0, 3.0, 4.0.... After the changes to the study protocol are approved by the certified clinical research review committee, the principal investigator submits a notification of changes to the implementation plan to the regional health bureau and also indicates the date of the certified clinical research review committee's approval on the cover page of the study protocol. After approval by the certified clinical research review committee, permission is obtained from the administrator of the implementing medical facility regarding the details of the amendment. If approval is granted, the principal investigator sends a copy of the approval letter to the research office and the changes to the study protocol take effect (the principal investigator decides whether to suspend patient enrolment during this period). The research office will announce the actual effective date, and after the effective date, the study will be conducted in accordance with the changes approved by the certified clinical research review committee. Until the effective date, the intervention and evaluation of enrolled patients will be conducted according to the version of the study protocol before the amendment, but if the safety of the subjects is threatened by the content of the study protocol before the change, such as inadequate criteria for changing the intervention, deviations from the study protocol to enhance the safety of the subjects during the intervention will be permitted. 15.6.2. Revisions of study protocol. 1) Do not increase the risk to study participants. WISH-J study version 1.5 4/23/2025 34 2) No substantial effect on the study's primary outcome. 3) Do not essentially affect the conduct of the study. Changes to a study protocol that meet all of the above are defined as revisions. Including changes due to typographical errors or facility-specific information changes, study protocol changes not involving study protocol changes regarding facility-specific information, and conflict of interest changes. Patient registration will not be temporarily suspended in the event of a revision. Version numbers of the study protocol and the informed consent document due to revisions are shown to one decimal place, e.g. 1.1, 1.2, 1.3.... When the changes to the study protocol are approved by the certified clinical research review committee, a notification of changes to the implementation plan is submitted to the regional health authorities and the date of approval by the certified clinical research review committee is noted on the cover page of the study protocol. The effective date of the changes to the study protocol are after the jRCT publication and the facility manager's approval. The effective date is the date after the submission of the notification of changes to the implementation plan to the regional health authorities. The actual effective date will be announced by the research office and the study will be conducted after the effective date in accordance with the revised content approved by the certified clinical research review committee. If the changes affect the format of the electronic case report form (eCRF), the principal investigator will request the data management officer to change the format. 15.6.3. Subject explanation and re-consent for study protocol changes When study protocols are changed, principal investigators correspondingly change subject informed consent documents. When certified clinical research review committees provide opinions that written subject explanation and re-consent are necessary, written consent is obtained again. 15.7. Reference 1) Lack G. Epidemiologic risks for food allergy.J Allergy Clin Immunol2008;121 :13311336 . 2) Natsume O., et al. Two-step egg introduction for prevention of egg allergy in high-risk infants with eczema (PETIT): a randomised, double-blind, placebocontrolled trial.Lancet2017;389 :276-286 . 3) Du Toit G., et al. Randomized trial of peanut consumption in infants at risk for peanut allergy.N Engl J Med2015;372 :803-813 . 4) Sakihara T., et al. Randomized trial of early infant formula introduction to prevent cow's milk allergy.J Allergy Clin Immunol2021;147 : 224-232.e228. 5) Perkin M. R., et al. Randomized Trial of Introduction of Allergenic Foods in Breast-Fed Infants.N Engl J Med2016;374 :1733-1743 . 6) Nishimura T., et al. Early introduction of very small amounts of multiple foods to infants: A randomized trial. Allergol Int2022;71 :345-353 . 7) Yasudo H., et al. Association of walnut proteins in household dust with household walnut consumption and Jug r 1 sensitization. Allergol Int2023;72 :607-609 . 8) Snetselaar L. G., et al. Dietary Guidelines for Americans, 2020-2025: Understanding the Scientific Process, Guidelines, and Key Recommendations. Today2021;56 :287-295 . 9) Leung J., et al. Increased Rates of Peanut and Tree Nut Aspiration as a Possible Consequence of Allergy Prevention by Early Introduction. Immunol Pract2021;9 : 31403146.e3142. 10) Quake A. Z., et al. Early Introduction of Multi-Allergen Mixture for Prevention of Food Allergy: Pilot Study. nutrients2022;14 :. 11) Quake A. Z., et al. Correction: Quake et al. Early Introduction of Multi-Allergen Mixture for Prevention of Food Allergy: Pilot Study. Nutrients 2022, 14, 14,. 737.Nutrients2022;15 :. WISH-J study version 1.5 4/23/2025 35 12) McWilliam V. L., et al. The TreEAT trial: protocol for a randomized controlled trial investigating the efficacy and safety of early introduction of tree nuts for Pediatr Allergy Immunol2023;34 : e13930. 13) Tariq S. M., et al. Cohort study of peanut and tree nut sensitisation by age of 4 years.Bmj1996;313 :514-517 . 14) Somani V. K. A study of allergen-specific IgE antibodies in Indian patients of atopic dermatitis.Indian J Dermatol Venereol Leprol2008;74 :100-104 . 15) Ruiz Segura L. T., et al. Food allergen sensitization patterns in a large allergic population in Mexico. Allergol Immunopathol (Madr)2020;48 :553-559 . 16) Moghtaderi M., et al. Specific IgE to common food allergens in children with atopic dermatitis.Iran J Immunol2012;9 :32-38 . 17) Kahveci M., et al. Immunoglobulin E-Mediated Food Allergies Differ in East Mediterranean Children Aged 0-2 Years. Int Arch Allergy Immunol 2020;181 :365-374 . 18) Lee J., et al. Component resolved diagnosis of walnut allergy in young children: Jug r 1 as a major walnut allergen. Asian Pac J Allergy Immunol2021;39 :190-196 . 19) Miyaji Y., et al. Effectiveness and safety of low-dose oral immunotherapy protocols in paediatric milk and egg allergy. 20) Sampson H. A., et al. Standardizing double-blind, placebo-controlled oral food challenges: American Academy of Allergy, Asthma & Immunology-. European Academy of Allergy and Clinical Immunology PRACTALL consensus report. J Allergy Clin Immunol2012;130 :1260-1274 . 15.8. appendix 1) Attachment 1: Response flow for the appearance of allergic symptoms due to ingestion of test foods 2) Exhibit 2: Diagnostic Definition of Food Protein-Induced Gastroenteropathy 3) List of Research Physicians 4) Procedures for Reporting Diseases, etc. 5) Monitoring Procedures and Plans 6) Logbook_WISH-J 7) Requirements for Implementing Medical Institutions *In addition, specifications and notes for 1) and 6), which are materials for participants in the Appendix, may be adjusted as appropriate during operation in consideration of ease of understanding, etc.