Cow's milk and egg protein threshold dose distributions in children tolerant to beef, baked milk, and baked egg

Abstract Background The use of eliciting doses (EDs) for food allergens is necessary to inform individual dietary advice and food allergen risk‐management. The Eliciting Dose 01 (ED01) for milk and egg, calculated from populations of allergic subjects undergoing oral food challenges (OFCs), are 0.2 mg total protein. The respective Eliciting Dose 05 (ED05) is 2.4 mg for milk and 2.3 mg for egg. As about 70% children allergic to such foods may tolerate them when baked, we sought to verify the EDs of that subpopulation of milk and egg‐allergic children. Methods We retrospectively assessed consecutive OFC for fresh milk and egg between January 2018 and December 2020 in a population of baked food‐tolerant children. Results Among 288 children (median age 56 ‐ IQR 36–92.5 months, 67.1% male) included, 87 (30.2%) returned positive OFC results, 38 with milk and 49 with egg. The most conservative ED01 was 0.3 mg total protein (IQR 0.03–2.9) for milk and 14.4 mg total protein (IQR 3.6–56.9) for egg. The respective ED05 was 4.2 (IQR 0.9–19.6) mg for milk and 87.7 (IQR 43–179) mg for egg. Such thresholds are, respectively, 1.5 (milk ED01), 1.75 (milk ED05), 72 (egg ED01), and 38.35 (egg ED05) times higher than the currently used thresholds. Conclusions The subpopulation of children allergic to milk and egg, but tolerant to baked proteins, displays higher reactivity thresholds than the general population of children allergic to milk and egg. Their risk stratification, in both individual and population terms, should consider this difference. In baked milk‐tolerant children, milk causes reactions at lower doses than egg in our group of egg‐tolerant children. This could be associated with the relative harmlessness of egg compared with milk in the determinism of fatal anaphylactic reactions in children.


| INTRODUC TI ON
Allergic reactions to milk and egg proteins are the most prevalent food allergies in European children. 1,2 They are a common cause of severe life-threatening reactions in pediatric allergic patients and could persist throughout life, placing a heavy burden on the quality of life, the development of other allergic diseases, and growth. [3][4][5] In the most sensitive individuals, trace amounts of milk and egg allergens may provoke allergic reactions, even from incidental contacts as opening packages, inhaling vapors from cooking, use of shared utensils, and kissing the lips of someone who has eaten the offending food. 6,7 Such reactions may be life-threatening, in particular for cow's milk. This food triggers more than 25% cases of fatal anaphylaxis in children, while egg has only exceptionally been associated with mortality for food allergy. 8 The primary and safest strategy for managing milk/egg allergy is strict avoidance of the causal food. To implement it, clinicians personalize their advices to patients informing the suggestions to their threshold of reactivity at oral food challenges (OFCs) among other clinical considerations. The population of milk-and eggallergic children includes a spectrum of more and less sensitive individuals. The former may also react to milk and egg in baked products as muffins or biscuits; less sensitive children tolerate baked foods. 9 In addition, the 10-20 percent milk-allergic children reacting to beef are considered having a more severe and persistent Cow's Milk Allergy (CMA). 10,11 When OFC-negative to baked milk and egg, children are not requested to avoid such foods. 12 Foodallergic children with a high reactivity threshold may not be requested to avoid precautionary-labelled prepackaged foods. 13,14 The use of precautionary allergen labelling (PAL) is based on population-based threshold dose distributions for the different food allergens. 15,16 To this end, data from cohorts of subjects assessed in oral food challenges (OFCs) are used to calculate eliciting doses (EDs) and to specify the relation between EDs and symptoms. 17,18 Based on such thresholds, the Voluntary Incidental Trace Allergen Labelling (VITAL), a scheme developed by the Australian and New Zealand food industries, established reference doses of 0.2 mg total protein (Eliciting Dose 01) for milk and egg, 16,19 and an Eliciting Dose 05 between 0.5 and 2.4 mg protein for both foods. 16,20,21 Such thresholds have been calculated on populations of patients allergic to both raw and baked foods. As raw and baked food are not likely equivalent, we aimed to explore the EDs of the subset of children allergic to milk and egg, who tolerate beef and baked proteins. We posed the hypothesis that children allergic to native products, but not to baked foods, may have a higher reactivity threshold that may exempt them from compliance with the current PAL.

| Study design
At the Bambino Gesù Allergy Department, patients with suspected milk/egg allergy are periodically exposed to oral food challenges (OFCs) until they get tolerance. For safety reasons, patients are excluded from OFC if an anaphylactic reaction had occurred in the last 6 months for children of 0.5-5 years, 12 months for children of 6-12 years, and 2 years for patients aged over 13 years. 22 Also not In this setting, we retrospectively assessed consecutive children who underwent OFC for fresh milk and egg between January 2018 and December 2020. For those who underwent repeated OFCs to the same food, we included only the first procedure for each type of food.

G R A P H I C A L A B S T R A C T
The clinical suspicion was placed based on the clinical history, and the entry symptoms were classified on parents' descriptions reported in the personal medical records. We only considered children with a personal history of acute IgE-mediated allergic reactions (developing within 1-2 h after food intake) from mild-to-severe systemic reactions (anaphylaxis). Before the OFC, parents were exhaustively informed about the risks of the procedure and gave their written consent. On the day, we administered the OFC, the sensitivity to the respective food was tested at skin prick test (SPT) and spe-

| Symptom grading and threshold data
Our trained staff performed OFCs in hospital with close supervision and immediate availability of emergency treatment, according to guidelines. The same clinicians observe and value the oral food challenges' result, and the doses were administered until objective symptoms appeared. 25,26 Briefly, the reactive symptoms classified patients in five groups, from subjective symptoms (nausea, abdominal pain, pruritus, oral allergy syndrome) to systemic reactions ( Table 2). 27 For this study, we designated the initial point at which objective symptoms occur at a specific dose as the Lowest Observed Adverse Effect Level (LOAEL) and the highest dose that does not lead to objective symptoms as the No Observed Adverse Effect Level (NOAEL).
According to previous studies, we choose an Interval-Censoring Survival Analysis (ICSA) approach to our data. This method is necessary when the exact dose that provokes a reaction in a patient is unknown, but it falls into a particular interval. Specifically, as previously described, 28 if a patient had an objective reaction at the first dose, left censoring occurred, and the NOAEL was set to zero with the LOAEL set as that first dose. If a patient did not experience an objective reaction after the 7th dose, the cumulative given dose was designated as NOAEL. In this case, we considered the data to be right-censored, and the LOAEL set to infinity. If the patient was unable or unwilling to continue the challenge because of the subjective symptoms, we discontinued the challenge and considered the final administered dose right censored. In all other cases, intervalcensoring occurs bounded by the NOAEL and LOAEL.

| Statistical analysis
We correlated the individual threshold to age and sex of patients, history of clinical manifestations, sIgE level results, or SPTs wheal size using multiple logistic regression (SPSS package, BM Corporation). A p-value of <.05 was considered statistically significant; sIgE values >100 kU/L received a designated value of 101 kU/L.
We also used SAS 15.1 (SAS Institute) and its LIFEREG procedure to fit parametric models to the interval-censored data. We considered Log-normal, log-logistic, and Weibull models to fit these data and to extrapolate EDs (ED01, ED05, ED10, ED25, and ED50), which are the doses predicted to elicit allergic reactions in 1%, 5%, 10%, 25%, and 50% of milk and egg-allergic patient, respectively. 29 The goodness-of-fit was assessed by the Anderson-Darling test.
Confidence intervals (CI) were calculated and added.
Step   Table S1 in this article's Online Repository).

| Eliciting doses
We fitted the data with log-normal, log-logistic, and Weibull distributions models (Figures 2A,B)

| DISCUSS ION
This is the first study aimed to evaluate the thresholds of children allergic to native egg and milk only. Our patients were tolerant to beef, baked milk, and baked egg. This population, which can be identified by specific challenges to baked foods, 30 represents the majority of milk/egg allergy sufferers. As they display a shorter duration of the condition, and present with less complex clinical pictures, they are considered those with the less severe forms of milk and egg allergy. 31 The prevalence of positive OFC in our center was 26.1% and 34.5% for milk and egg, respectively. While a similar prevalence was reported in children with OFCs positive to egg (33%) and milk (32%), 32 other caseloads showed higher prevalence rates, both for milk and egg. [33][34][35][36][37] Our threshold values are similar to those found in VITAL for milk  Table 2) VITAL, consistently with the fact that they are allergic to native egg only and that no statistical correlation between patients' individual threshold and their age, gender, sIgE, and/or SPT levels was found.
Our milk allergic children also showed a higher reactivity threshold than that of VITAL both at level 01 and at level 05, but differences were much smaller. Again, this is unlikely to be due to different food doses at challenge. Studies with milk OFCs in general use inferior cumulative amounts of milk, with starting doses similar [48][49][50] or higher, 51,52,53 to our 3 mg milk protein.
These findings echo those of other studies for milk, but not for egg. For instance, in a single-center pediatric experience using a cumulative dose amount of protein of 2190 mg, corresponding to about 63 ml of milk and 1/3 egg, the ED05 were set at 1.07 for milk and at 1.51 for egg. 54 The only study comparing thresholds of baked-tolerant vs. baked-sensitive egg-and milk-allergic patients found ED10 and ED50 values similar to ours for milk, but much lower for egg 55 (Table 4). We recently published tolerance threshold data of children with severe baked milk and baked egg allergy, using a different OFC scheme than the one used in this study. The mean thresholds of reactivity we found in that context were 116.3 (±107.6) and 128.3 (±96.7) mg protein for milk and egg, respectively; the EDs were not calculated. 22 A further study found much lower levels ED05 and ED10 for milk. 56 These differences could be due to different selected populations, difference in mean age, and difference in the method of administration of the OFC. In any case, should our results be confirmed by other studies they could explain why, in spite of a similar prevalence in the population, milk continues to be more represented than egg in the caseloads of mortality due to food anaphylaxis in children. 8,59 It is licit to infer that lower thresholds may induce parallel higher probability of severe reactions. Despite their low ED01, we found a low frequency In most studies, EDs for cow's milk and egg proteins have been associated to the age of patients, the severity of allergic reactions or the OFC protocol used, such as the number of doses and the total amount of food allergen administered. 32,33,63,64 Therefore, risk analysis for specific groups of patients (i.e., children vs. adults, comparison between various countries) had statistical limits which may influence results when comparing retrospective data from single studies or single protocol-specific datasets. 16,54 In conclusion, our study found similar milk EDs, but higher egg EDs compared with those previously found in other studies. Our data suggest that children with baked egg tolerance may be less exposed to the risk of severe reactions than children with baked milk tolerance.
They could be tolerating to egg traces and perhaps they could assume egg-PAL pre-packaged foods. Should our results be confirmed by studies comparing EDs differences between raw and baked food in pediatric population, the clinical attitude toward the two allergies could be differentiated. To confirm these data, we will evaluate if such difference in thresholds is present also in children allergic to baked egg/milk. For the moment, these data add to the knowledge about thresholds of reactivity for these foods, an essential characteristic of the hazard that allergens present to the food-allergic population. 65

Open access funding provided by Ospedale Pediatrico Bambino
Gesu.

AUTH O R CO NTR I B UTI O N S
RLV developed the concept, prepared the infrastructure of the study, elaborated the data, performed the statistical analysis, and contributed made the draft. AF initiated the concept and contributed made the first draft. CR, VC, LD, VF, MM, and AS administered the OFCs, evaluated the clinical data, included them in the database and contributed to the discussion. ALP collaborated to the inclusion of data in the database, collaborated to the statistical analysis, and contributed to the discussion. All authors reviewed and approved the final manuscript.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflicts of interest to disclose. Note: ED01, ED05, ED10, ED50: cumulative amount of food protein predicted to cause an allergic reaction in 1%, 5%, 10%, or 50% of the food allergic population, respectively.