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June 2005

# Approaches to Establish Thresholdsfor Major Food Allergens and for Gluten in Food

## DRAFT REPORTPrepared byThe Threshold Working Group

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## Table of Contents

      - Executive Summary
      - Acknowledgements
      - Overview          - Purpose
        - Definitions of Thresholds
        - FALCPA Requirements
      - Food Allergy          - Food Allergy and Food Intolerance
        - Mechanism of Allergic Reaction
        - Range of Adverse Effects
        - Prevalence
        - Allergenic Foods of Concern
        - Measuring Thresholds
        - Exposure
        - Collective Allergens
        - Published Challenge Studies
        - Food Treatments to Reduce Allergenicity
      - Celiac Disease          - Introduction
        - Mechanism of Pathogenesis
        - Range of Adverse Effects
        - Prevalence
        - Celiac Foods of Concern
        - Gluten Contamination of Grains
        - Gluten Challenge Studies
        - Measuring Gluten in Food
        - Gluten-Free Labeling
      - Discussion and Recommendations          - General Approaches
        - General Criteria for Evaluating and Selecting Approaches to Establish Thresholds
        - Allergen Thresholds: Evaluation and Findings
        - Gluten Threshold: Evaluation and Findings
      - References
      - Appendices          - Appendix 1:  Evaluation of Analytical Methods for Food Allergens
        - Appendix 2:  Evaluation of Available Allergen Oral Challenge Studies
        - Appendix 3:  Evaluation of Published Measurements of Protein Concentrations in Oils
        - Appendix 4:  Evaluation of Gluten Testing Methods
        - Appendix 5:  Evaluation of Gluten Oral Challenge Studies
      - List of Tables         - Table I-1.  Summary of Various Types of Thresholds
        - Table II-1.  Symptoms of Allergic Reactions
        - Table II-2.  Allergy Prevalence in the United States
        - Table III-1.  Estimated Daily Gluten Consumption from Combinations of Different Amounts of Food Containing Different Levels of Gluten
        - Table IV-1.  Approaches to Establishing Thresholds
        - Table IV-2.  General Criteria for Evaluating and Selecting Recommended Approaches to Establish Thresholds
        - Table IV-3.  Specific Criteria for Evaluating Analytical Methods for Food Allergens
        - Table IV-4.  Specific Criteria for Evaluating Allergen Oral Challenge Studies
        - Table IV-5.  Summary of Published LOAELs for Food Allergens
        - Table IV-6.  Example of Uncertainty Factors for the Safety-Assessment-Based Approach Using Peanuts
        - Table IV-7.  Specific Criteria for Evaluating Protein in Oil Studies
        - Table IV-8.  Specific Criteria for Evaluating Gluten Analytical Methods
        - Table IV-9. Specific Criteria for Evaluating Gluten Oral Challenge Studies
        - Table IV-10. Example of Uncertainty Factors for the Safety-Assessment-Based Approach
      - List of Figures         - Figure II-1.  Food Intolerance and Hypersensitivity
        - Figure II-2.  Mechanism of Allergic Reactions
        - Figure III-1 Mechanism of Celiac Disease

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## Executive Summary

### Background

This report summarizes the current state of scientificknowledge regarding food allergy and celiac disease, including information ondose-response relationships for major food allergens and for gluten,respectively.  The report presents the biological concepts and data needed toevaluate various approaches to establishing thresholds that would be scientificallysound and efficacious in relation to protection of public health.  Eachapproach has strengths and weaknesses, and the application of each is limitedby the availability of appropriate data.  It is likely that there will besignificant scientific advances in the near future that will address a numberof the limitations identified in this report.

The Threshold Working Group expects that any decisions onapproaches for establishing thresholds for food allergens or for gluten wouldrequire consideration of additional factors not covered in this report.Furthermore, one option that is implicit in the report's discussion ofpotential approaches is a decision not to establish a threshold at this time.

### Approaches to Establish Thresholds

The report identifies four approaches that could be used to establish thresholds:
      - Analytical methods-based -thresholds are determined by the sensitivity of the     analytical method(s) used to verify compliance.
      - Safety assessment-based -a "safe" level is calculated using the No Observed     Adverse Effect Level (NOAEL) from available human challenge studies and an     appropriate Uncertainty Factor applied to account for knowledge gaps.
      - Risk assessment-based -examines     known or potential adverse heath effects resulting from human exposure to     a hazard; quantifies the levels of risk associated with specific exposures     and the degree of uncertainty inherent in the risk estimate.
      - Statutorily-derived -uses     an exemption articulated in an applicable law and extrapolates from that to     other potentially similar situations.  

### Threshold Working Group Findings For Major Food Allergens

Finding 1. The initial approach selected to establishthresholds for major food allergens, the threshold values, and any uncertaintyfactors used in establishing the threshold values should be reviewed andreconsidered periodically in light of new scientific knowledge and clinicalfindings.

Finding 2. The **analytical methods-based approach**can be used to establish thresholds for those major food allergens for whichvalidated analytical methods are available.  However, if this approach is used,the thresholds should be replaced by thresholds established using one of theother approaches as quickly as possible.

Finding 3.  The **safety assessment-based approach**,based on currently available clinical data, is a viable way to establishthresholds for the major food allergens.  If this approach is employed, the LowestObserved Adverse Effect Level (LOAEL) or No Observed Adverse Effect Level (NOAEL)determinations used should be based on evidence of the "initial objectivesymptom."  Individual thresholds should be established for each of the major foodallergens.  If it is not feasible to establish individual thresholds, a singlethreshold based on the most potent food allergens should be established.  Inthose instances where a LOAEL is used rather than a NOAEL to establish athreshold, an appropriate uncertainty factor should be used.

Finding 4.  Of the four approachesdescribed, the quantitative **risk assessment-based** approach provides thestrongest, most transparent scientific analyses to establish thresholds for themajor food allergens.  However, this approach has only recently been applied tofood allergens, and the currently available data are not sufficient to meet therequirements of this approach.  A research program should be initiated todevelop applicable risk assessment tools and to acquire and evaluate theclinical and epidemiological data needed to support the quantitative riskassessment-based approach.  Thresholds established using this approach shouldbe reevaluated periodically as new data and tools become available.

Finding 5.The **statutorily-derived approach**provides a mechanism for establishing thresholds for allergenic proteins infoods based on a statutory exemption.  Potentially, this approach could be usedto set a single threshold level for proteins derived from any of the major foodallergens.  This approach might yield thresholds that are unnecessarily protectiveof public health as compared with thresholds established using the safetyassessment-based approach.  However, confirming this would require additionaldata.  If this approach is employed to establish thresholds, it should be usedonly on an interim basis and should be reevaluated as new knowledge, data, andrisk assessment tools become available.

### Threshold Working Group Findings For Gluten

Finding 6.The initial approach selected to establish athreshold for gluten, the threshold value selected, and any uncertainty factorsused to establish the threshold should be reviewed and reconsidered periodicallyin light of new scientific knowledge and clinical findings.

Finding 7.  The **analytical methods-based approach**can be used to establish a threshold for gluten.  However, if this approach isused, the threshold should be replaced by a threshold established using one ofthe other approaches as quickly as possible.

Finding 8.The **safety assessment-based approach**is a viable approach to establish a threshold for gluten using currentlyavailable LOAEL data for celiac disease.  An overall uncertainty factor shouldbe estimated from the data and applied to the LOAEL to establish a thresholdfor gluten.  Any threshold derived from this approach should be reevaluated asnew research data become available.  Available data are insufficient at thecurrent time to use this approach to establish a threshold for oat gluten forthose individuals with celiac disease who are also sensitive to oats.  However,it is likely that a threshold based on wheat gluten would be protective forindividuals susceptible to oat gluten.

Finding 9.Use of the quantitative **riskassessment-based approach** to establish a threshold for gluten does notappear to be feasible at the present time.  However, considering the benefitsthat could be gained from using the risk assessment-based approach, priorityshould be given to establishing a research program to acquire the knowledge anddata needed.

Finding 10. There appear to be no suitable legalrequirements or exemptions that would serve as the rationale using for a **statutorily-derived approach** to establish a threshold for gluten.  Thisapproach is not viable.

Any approach used to establish a threshold to protectconsumers with food allergies or susceptible to celiac disease should be usedin an iterative manner.  The threshold approach should be re-examinedperiodically to consider new knowledge, data, and approaches.

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### Acknowledgements

Threshold Working GroupRobert Buchanan (Lead)Sherri Dennis (Associate Lead)

Core Writing TeamSteven Gendel (Lead)David CarlsonSherri Dennis

Working Group MembersDavid AchesonSue Anne AssimonNega BeruPhilip BolgerRicardo CarvajalCatherine CoppKenneth FalciElizabeth HardenRhonda KaneJohn KvenbergStefano LuccioliDouglas ParkRichard RaybourneTerry TroxellKatherine Vierk

The Threshold Working Group would also like to acknowledge the assistance provided by:

Lori Pisciotta in preparation of the report,Eric Garber for information on methods of analysis,Curtis Barton for information onstatistics and study designs.Michael Landa for consultations on the FALCPA.

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## I. Overview

### A. Purpose

Accurate and informative labeling is critical for allergicconsumers, individuals with celiac disease, and their families because theyneed to rely on strict avoidance to prevent potentially serious reactions.  TheFood Allergen Labeling and Consumer Protection Act of 2004 (P.L. 108-282)(FALCPA) amends the Federal Food, Drug, and Cosmetic Act (FFDCA) and requiresthat the label of a food product that is or contains an ingredient that bearsor contains a "major food allergen" declare the presence of theallergen as specified by FALCPA.  FALCPA defines a "major foodallergen" as one of eight foods or a food ingredient that contains proteinderived from one of those foods.

An important scientific issue associated with theimplementation of FALCPA is the existence of threshold levels below which it isunlikely that a food allergic individual would experience an adverse effect.FALCPA provides two processes by which an ingredient may be exempted from theFALCPA labeling requirements, a petition process (21 U.S.C. 343(w)(6)) and anotification process (21 U.S.C. 343(w)(7).)  Under the petition process, aningredient may be exempt if the petitioner demonstrates that the ingredient"does not cause an allergic reaction that poses a risk to human health."  Underthe notification process, an ingredient may be exempt if the notificationcontains scientific evidence that demonstrates that the ingredient "does notcontain allergenic protein," or if FDA previously has determined, under section409 of the FFDCA, that the food ingredient does not cause an allergic responsethat poses a risk to human health.  Thus, understanding food allergenthresholds and developing a sound analytical framework for such thresholds arelikely to be centrally important to FDA's analysis of, and response to, FALCPApetitions and notifications.

FALCPA also requires FDA to promulgate a regulation to define and permit the use of the term "gluten free" on the labeling of foods.  Such labeling is important to patients suffering from celiac disease, an immune-meditated illness.  Strict avoidance of gluten at levels that will elicit an adverse effect is the only means to prevent potentially serious reactions.  Thus, consumers susceptible to celiac disease need accurate, complete, and informative labels on food to protect themselves. Understanding thresholds for gluten will help FDA develop a definition of "gluten free" and identify appropriate use of the term. Section 204 of FALCPA directs FDA to prepare and submit a report to Congress.  The report is to focus principally on the issue of cross-contact of foods with food allergens, and is to describe the types, current use of, and consumer preferences with respect to advisory labeling.  Cross-contact may occur as part of the food production process where residues of an allergenic food are present in the manufacturing environment and are unintentionally incorporated into a food that is not intended to contain the food allergen, and thus, the allergen is not declared as an ingredient on the food's label.  In some cases, the possible presence of the food allergen is declared by a voluntary advisory statement.  Understanding food allergen thresholds and developing a sound analytical framework for such thresholds is also likely to be useful in addressing food allergen cross-contact and the use of advisory labeling.Both as part of its on-going risk management of food allergens and in response to FALCPA, CFSAN established an internal, interdisciplinary group (the Threshold Working Group) to evaluate the current state of scientific knowledge regarding food allergies and celiac disease, to consider various approaches for establishing thresholds for food allergens and for gluten, and to identify the biological concepts and data needed to evaluate the scientific soundness of each approach.  This draft report is the result of the working group's deliberations.

This draft report summarizes the current state of scientificknowledge regarding food allergies and celiac disease, including information ondose-response relationships for major food allergens and for gluten,respectively.  The ability to establish a threshold depends on understandingthe dose-response relationship between the ingestion of an allergen or glutenand the elicitation of an adverse response.  Implicit in establishing suchdose-response relationships is the identification of susceptible populationsand characterization of any threshold levels below which all, or part, of thesusceptible population does not respond.  This draft report identifies thebiological concepts and data needed to evaluate various approaches forestablishing thresholds that would be scientifically sound and efficacious inrelation to protection of public health.

### B. Definitions of Thresholds

The term "threshold" has been used to refer to a variety ofdifferent concepts (Table I-1) that apply either to individuals orpopulations.  Thresholds can be measured experimentally in animals or humans [i.e.,No Observed Adverse Effect Level (NOAEL) or Lowest Observed Adverse EffectLevel (LOAEL)], derived from epidemiological data, estimated by modeling(statistical or simulation), established by statute, or arise as the result ofthe selection of an analytical method.  The ability to measure or determine athreshold may be limited by the sensitivity and specificity of the methodsavailable to measure either the stimulus or the response.  Understanding thestrengths and limitations of the data underpinning the different approaches isparticularly important when dealing with adverse effects that have lowprobabilities of occurring.
| Type                    | Description                                                                                                                                     |
|-------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------|
| Etymological Definition | "The intensity below which a mental or physical stimulus  cannot be perceived and can produce no response."  (Webster's Dictionary). |
| Toxicological           | The dose at, or below which, an adverse effect is not seen  in an experimental setting.                                                        |
| Methodological          | The limit of detection of an analytical method.                                                                                                 |
| Statutory               | The establishment of a limit by statute, below which no  regulatory action will be taken.                                                      |

### C. FALCPA

As noted, FALCPA amends the FFDCA to prescribe the manner inwhich food labels must disclose that a food is, or contains an ingredient thatbears or contains, a major food allergen.  The law also requires the FDA toissue a regulation to define and permit use of the term "gluten-free."   

FALCPA establishes a petition process through which a foodingredient may be exempt from FALCPA's labeling requirements if the ingredientdoes not cause an allergic response that poses a risk to human health.  FALCPAalso establishes a notification 

process under which a food ingredient described in section201(qq)(2) of the FFDCA may be exempt from FALCPA's labeling requirements ifthe ingredient does not contain allergenic protein, or if FDA previously hasdetermined, under section 409 of the FFDCA, that the food ingredient does notcause an allergic response that poses a risk to human health.

From the perspective of the Working Group, implementation ofthe FALCPA petition and notification provisions could present several keyscientific issues.  First, what is an "allergic response?"  Second, do all allergicresponses pose a risk to human health, or do some allergic responses pose moreof a risk than others?  Third, can allergens occur in a food either in a formor at a level that is too low to cause harm (i.e., either the allergen does notcause a biological response or the response is too mild to be consideredhazardous)?

Under FALCPA, a "highly refined oil" derived from one ofeight foods or food groups and "any ingredient derived from such highly refinedoil" are exempt from the definition of "major food allergen" and fromFALCPA's labeling requirements.  As discussed further below, there is evidencethat consumption of highly refined oils does not appear to be associated withallergic responses despite the potential presence of low levels of protein inthese oils.

Section 206 of FALCPA requires FDA to issue a proposed ruleto define and permit use of the term "gluten-free" on labeling of foods.Section 203 of FALCPA recognizes that"the current recommended treatment forceliac disease is avoidance of glutens in foodsthat are associated with" thedisease.  FALCPA does not directly state how the term "gluten-free"should be defined.II. Food AllergyA. Food Allergy and Food Intolerance
Many consumers consider a wide variety of adverse effectsassociated with the ingestion of foods to be "food allergies."  These adverse effectsmay occur for a variety of immunologic, toxicologic, or metabolic reasons.  Thesymptoms associated with these effects can range from oral irritation andsensitivity, to enteropathies (gastrointestinal tract injury, pain, andnutrient malabsorption), colitis, and eczema (Jackson, 2003).  In someinstances, these symptoms can be caused by toxic compounds such as histamine,which is formed by microbial conversion of naturally occurring histidine infoods.  In other instances, adverse effects can be caused by metabolicconditions such as lactose intolerance.  While these conditions are welldocumented and in some cases potentially life threatening, they are mostappropriately termed food intolerances (Figure II-1) (Johansson *et al*.,2001; Sampson, 2004).

Immune responses to components of foods can occur that adverselyaffect portions of the population.  These immune responses include: (1) immunoglobulinE (IgE)-mediated hypersensitivity (e.g., oral allergy syndrome, anaphylaxis),(2) cell-mediated hypersensitivity (e.g., celiac disease, food protein-inducedenterocolitis), and (3) combined IgE- and cell-mediated immunity (e.g., eosinophilicgastroenteritis, atopic dermatitis).  For the purposes of this report, the term"food allergy" will be used to describe IgE-mediated immune responses resultingfrom the ingestion of specific foods (Johansson *et al*., 2001; Jackson,2003; Sampson, 2004).  The most severe and immediately life-threatening adverseeffects are associated with IgE-mediated hypersensitivity (Johansson *et al*.,2001; Jackson, 2003; Zarkadas *et al*., 1999).

**Figure II-1.  Food Intolerance andHypersensitivity**
B. Mechanism of Allergic Reaction
An allergic reaction stems from an abnormal, or exaggerated,immune system response to specific antigens, which in foods are proteins (Sampson,1999).  This immune response occurs in two phases, an initial "sensitization"to an allergen and the "elicitation" of an allergic reaction on subsequentexposure to the same allergen.  Sensitization occurs when a susceptibleindividual produces IgE antibodies against specific proteins in a food.  Upon re-exposureto the same food allergen, the allergenic proteins bind to IgE molecules onimmune mediator cells (basophiles and mast cells), leading to activation ofthese mediator cells.  This elicitation causes the release of inflammatorymolecules (e.g., leukotrienes and histamine).  The specific symptoms andseverity of an allergic reaction are affected by the concentration of allergen,route of exposure, and the organ systems involved (e.g., skin, GI tract,respiratory tract, and blood) (Taylor and Hefle, 2001).

**Sensitization**

**Elicitation**

**Figure II-2.  Mechanism of Allergic Reactions**
C. Range of Adverse Effects
The clinical manifestations of food allergic reactions rangefrom mild irritation to severe, life-threatening respiratory distress and shock.Specific symptoms may involve the skin (e.g., pruritis, erythema, urticaria,angiodemia, eczema), eyes (e.g., conjunctivitis, periorbital swelling), nose(e.g., rhinitis, sneezing), oral cavity (e.g., swelling and itching of lips,tongue, or palate), or gastrointestinal tract (e.g., reflux, colic, abdominalpain, nausea, vomiting, diarrhea).  In more severe reactions, involvement ofthe respiratory tract (e.g., cough, asthma, difficulty breathing, swellingaround the larynx and vocal cords) and cardiovascular system (e.g., faintness,hypotension) can lead to loss of consciousness, asphyxiation, shock, or death.The term "anaphylaxis" is used to describe multisystemic severe reactions to anallergen requiring immediate medical intervention (Jackson, 2003).

Table II-1 provides a summary of subjective and objectivesymptoms that can be experienced during an allergic reaction.  Allergicreactions usually occur within a few minutes to one hour after ingestion of anoffending food and often progress from mild to severe, with higher dosescausing more severe reactions (Sampson, 2005).  Once exposure occurs,individuals may experience immediate numbness or pruritis at the site ofcontact or experience general uneasiness.  These symptoms are characterized as"subjective" since they cannot be observed by others.  As the effects progress,"objective" symptoms such as flushed skin, hives, or swelling of the lips andface occur.  These symptoms are often mild and short-lived.  However, in somecases, they may be associated with more severe symptoms, involving the respiratoryand cardiovascular systems.  Such symptoms can lead to hospitalization or death,even with appropriate medical intervention.  Not all severe, or anaphylactic,reactions are necessarily preceded by milder signs and not all reactions areimmediate.  In some cases, anaphylactic reactions may be delayed by a few hoursafter initial symptoms (Sampson, 2005).

The severity of an allergic reaction is affected by severalfactors that include genetic predisposition (atopy), age, type of foodallergen, nature of any food processing, environment, and physiologicalconditions (Taylor and Hefle, 2001; Sampson, 2003; Maleki, 2004).  For example,exercise, medications (e.g., non-steroidal anti-inflammatories), alcoholconsumption, and asthma may enhance the severity of an allergic reaction(Sampson, 2005).  Most severe and fatal allergic reactions to foods haveoccurred in adolescents and teens who were highly atopic and had a history ofasthma (Sampson, 2003; Pumphrey, 2004).

It is generally assumed that a history of previous seriousallergic reaction(s) indicates an increased risk of severe reaction(s).However, a history of mild reactions does not preclude the possibility of asubsequent severe reaction.  For example, Sicherer *et al*. (1998)observed that mild reactions to peanut in childhood tend to become more severeand unpredictable in later childhood and adulthood.  This may be due to thefact that these children develop asthma later in life (Sampson, 2005).  Also, arecent review of anaphylactic fatalities in the United Kingdom showed that, in85% of fatal food reactions the patient had previously experienced a non-severereaction (Pumphrey, 2004).  Pumphrey (2004) states that the severity ofprevious reactions is not a risk factor for a fatal reaction for nut allergicpatients.  These data imply that any individual with a clinical history ofIgE-specific food allergy may be considered to be predisposed to anaphylaxis orsevere reaction.
Table II-1. Symptoms of Allergic Reactions  Subjective Symptoms Objective SymptomsaCUTANEOUS
Skin

Pruritus (Itching)
  Skin flushing or erythema (redness)  Pilor erection ("goosebumps")  Rash (hives) - acute  Eczema (usually delayed, >6 hours)  Angioedema (swelling, especially face)
Oral cavity (lips, tongue, palate)

Pruritus (Itching), numbness, dryness

Edema (swelling, may also include the uvula)

Eyes, conjunctiva

Pruritus (Itching)

Periorbital (around eyes) edema, redness of conjunctiva  and tearing 

GASTROINTESTINAL

Nausea, pain (except infants/young child)

Vomiting, diarrhea, abdominal pain (infants)
RESPIRATORY
Nose

Pruritus (Itching)

Nasal congestion or runniness, sneezing

Larynx, throat

Pruritus (Itching), dryness/tightness

Swelling around the larynx and vocal cord, voice  hoarseness, stridor (inspiratory wheeze), cough

Lungs

Shortness of breath, chest pain/tightness

Respiratory distress (i.e., ↑  breathing rate, difficulty catching breath, ↓  peak expiratory flow measurement), cough, wheezing

HEART and CARDIOVASCULAR

Chest pain/ tightness, feeling of faintness, dizziness

Syncope (fainting, loss of consciousness), hypotension  (low) or shock (very low blood pressure), dysrhythmia (abnormal heart rhythm)

OTHER

"Sense of impending doom"b

Uterine contractions (women)

a Anaphylaxis is a poorly defined condition representing a severe or multisystemic allergicreaction.  Allergic reactions described by objective symptoms involving therespiratory or cardiovascular systems would be considered severe and managed asan anaphylactic reaction by most clinicians.  In some classifications, symptomsin two or more categories above (e.g., cutaneous, gastrointestinal, respiratory),even if relatively mild, would also be classified as anaphylaxis.  Anaphylactic"shock"  denotes a consequence of anaphylaxis where heart irregularities andleakage of blood vessels leads to extreme blood volume loss (usually greaterthan 25% of resting blood volume) and extreme hypotension. 
b A "sense of impending doom" may signal or predict animpending severe reaction.
D. Prevalence
The most recent information on the prevalenceof food allergies in the U.S. suggests that up to 6% of children and 4% ofthe total population have IgE-mediated food allergies (Sampson, 1997; Sampson,2004; Sicherer *et al*., 2003; Sicherer *et al*., 2004).  Theestimated prevalence in the U.S. population of allergies to each of the foodallergens identified by the FALCPA is given in Table II-2.  Severe food-relatedallergic reactions result in an estimated 30,000 emergency room visits, 2,000hospitalizations, and 150 deaths per year (Sampson, 2004).  Clinical data andsurveys indicate that the prevalence of allergy has been rising in recentyears, though there are limited historical data to compare to recent numbers (Sicherer*et al*., 2003; Grundy *et al*., 2002).  Peanut allergy has receivedthe most attention in the U.S., and data indicate an apparent doubling ofpeanut allergy in children under 5 years old from 1997 to 2002 (Sicherer *etal*., 2003).  A similar increase in peanut allergy has been seen in the UnitedKingdom (Ewan, 1996; Grundy *et al*., 2002). Peanuts and tree nuts arethe most common cause for fatal reactions in the US (Yunginger *et al*.,1988; Sampson *et al*., 1992; Bock *et al*., 2001).
Table II-2.  Allergy Prevalence in the United  States  Age Group  Percentage of  the Population   All Allergens   Milk   Egg   Peanut   Tree nuts   Fish   Shellfisha  Wheat   Soy   Children (<3 years)   6.0   2.5   1.3   0.8   0.2   0.1   0.1   UNKb
0.2
  Adults (>18 years)   3.7   0.3   0.2   0.6   0.5   0.4   2.0   UNKb  0.2 
aShellfish includes both crustaceans and mollusks.  bUNK= unknown

Sources:  Sampson, 1997;Sampson, 2004; Sicherer *et al*., 2003; Sicherer *et al*., 2004.
E. Allergenic Foods of Concern
1.  Whole foods

The FALCPA identifies eight major food allergens or foodgroups:  milk, eggs, fish (e.g., bass, flounder, cod), crustacean shellfish(e.g., shrimp, crab, lobster), tree nuts (e.g., almonds, walnuts, pecans), peanuts,wheat, and soybeans.  These foods account for 90% of all documented IgE-mediatedfood allergies worldwide and most severe reactions to foods (Bousquet *et al*.,1998; Hefle *et al*., 1996).  More than 160 other foods are known to causefood allergies; however, these allergies are relatively rare with prevalencerates ranging from a few percent of the allergic population to single cases (Hefle*et al*., 1996).  Each of the eight major food allergens contains multipleallergenic proteins, many of which have not been fully characterized (Gendel,1998).

2.  Food Ingredients

Some food ingredients such as edible oils, hydrolyzedproteins, lecithin, gelatin, starch, lactose, flavors, and incidental additives(e.g., processing aids), may be derived from major food allergens (Taylor andHefle, 2001).  The role that these ingredients play in food allergy has notbeen fully characterized.  For example, lecithin is a common food ingredientwhich is often derived from soybeans.  It is possible that soy lecithin, whichcontains residual protein, could elicit an allergic reaction in sensitiveindividuals (Muller *et al*., 1998; Gu *et al*., 2001).  Anotherexample is protein hydrolysate, which is often made from a major food allergen suchas soybeans, wheat, peanuts, whey, or casein. Extensively hydrolyzed proteinspresent only slight risk to allergic individuals, but partially hydrolyzedprotein ingredients can elicit allergic reaction (Bock and Atkins, 1989; Ellis *etal*., 1991; Saylor and Bahna, 1991; Kelso and Sampson, 1993; Niggemann *etal*., 1999).  For example, hot dogs formulated with partially hydrolyzedcasein have elicited allergic reactions in children allergic to cow's milk(Gern, *et al*., 1991; Kocabas and Sekerel, 2003).

Gelatins are ingredients derived from animals (e.g., cows,pigs) but also from the skin of various species of fish. A study of 10 fishallergic patients and 15 atopic individuals with eczema revealed that 3 and 5individuals respectively had specific IgE to fish gelatin, suggesting thepresence of allergenic protein (Sakaguchi *et al*.,2000).  However, in a recent Double Blind PlaceboControlled Food Challenge (DBPCFC) study, all 30 fish allergic subjects in thestudy did not respond to a cumulative dose of 3.61 g of fish gelatin (Hansen *etal*., 2004).

Edible oils can be derived from major food allergens such assoybeans and peanuts, and they may contain variable levels of protein (Taylorand Hefle, 2001).  The consumption of highly refined oils derived from major foodallergens by individuals who are allergic to the source food does not appear tobe associated with allergic reactions.  For example, Taylor *et al*.(1981) and Bush *et al*. (1985) did not observe any reactions to refinedpeanut or soy oils in 10 and 7 allergic patients, respectively.  This may notbe the case for unrefined or cold-pressed oils that contain higher levels ofprotein residues (Taylor and Hefle, 2001).  For example, Hourihane *et al*.(1997) reported that 6 of 60 peanut allergic individuals reacted to crudepeanut oil but none responded to refined peanut oil.  Similarly, Kull *et al*.(1999) reported that 15 of 41 peanut allergic children responded positively tocrude peanut oil in skin prick tests, but none responded to refined peanutoil.  The actual protein levels reported in various edible oils varies,probably due to differences in the oil, refining process, and the proteindetection analytical method used.  Crevel *et al*. (2000) reported thatcrude peanut and sunflower oils contained 100-300 �g/ml of protein, but that the most highly refined oilscontained 0.2-2.2 �g/ml ofprotein.  Intermediate protein concentrations were seen for partially processedoils.  Teuber *et al*. (1997) showed that the amount of protein in bothcrude and refined gourmet nut oils varied both by type of oil and degree ofprocessing, and reported values of 10-60 �g/mlfor various unrefined oils and 3-6 �g/mlfor the refined oils.  Several other investigators reported undetectable levelsof proteins in refined edible oils (Hoffman *et al*., 1994; Yeung and Collins,1996; Peeters *et al*., 2004) using assays with detection sensitivities of<0.3 ng/ml (Peeters *et al*., 2004) and 0.4 mg/kg (Yeung and Collins,1996).

Starch, which is a widely used ingredient, is often derivedfrom corn which is not a major food allergen.  However, starch can also bederived from wheat, and may contain trace levels of wheat protein.  Most ofthese proteins are from the non-albumin fraction whereas the principal wheatallergenic proteins are albumins.  The allergenicity of wheat starch forsensitive individuals has not been clinically evaluated (Taylor and Hefle,2001).

A wide variety of flavoring substances are used in foods, butonly a few are derived from known allergens (Taylor and Dormedy, 1998).  Assuch, IgE-mediated allergic reactions to flavorings are rare, although a fewcases have been documented involving hydrolyzed proteins.  For example, severalmilk allergic individuals reacted to either hot dogs or bologna containingpartially hydrolyzed casein as part of the natural flavoring used in theformulation of these products (Gern *et al*., 1991).  Two othermilk-allergic individuals reacted to milk protein in the natural flavoring usedin a dill pickle-flavored potato chip (St. Vincent and Watson, 1994).  Thepresence of peanut flour in the natural flavoring of a packaged soup elicited areaction in a peanut-allergic individual (McKenna and Klontz, 1997).

3. Cross-Contact

Allergens, or proteins derived from allergenic foods, may bepresent in foods as the result of cross-contact during processing andhandling.  The term "cross-contact" describes the inadvertent introduction ofan allergen into a product that would not intentionally contain that allergenas an ingredient.  Cross-contact is generally the result of environmental exposureduring processing or handling, which may occur when multiple foods are producedin the same facility or on the same processing line, through the misuse ofre-work, as the result of ineffective cleaning, or from the generation of dustor aerosols containing the allergen.  Cross-contact of foods with allergens hasbeen shown to lead to allergic reactions in consumers on numerous occasions(Gern *et al*., 1991; Jones *et al*., 1992; Yunginger *et al*.,1983).  Much cross-contact can be avoided by controlling the production environment.Whether all cross-contact can be prevented during food manufacturing is yet tobe determined.
F. Measuring Thresholds
1. Design of Food Challenge Studies

A history of clinical reaction to a food and a positive skinprick test or the presence of food-specific IgE antibodies in serum aresufficient to establish that an individual has an allergy to that food.However, none of these reliably predicts the level of patient sensitivity.  Atpresent, individual sensitivity can only be determined using food challengestudies (including open, single-blind, and double-blind, placebo-controlledfood challenges).  The double-blind, placebo-controlled food challenge (DBPCFC)is the "gold standard" for diagnosis of food allergy and for determiningclinical reactivity to low concentrations of an allergen.  In these studies,neither the subjects nor the researchers know which test foods contain theallergen.  Open (where both the subjects and the researchers know which testfoods contain the allergen) and single-blinded (where only the researchers knowwhich foods contain the allergen) challenges are used primarily for screeningfoods of low allergenic importance or for determining tolerance to foodallergens.  Single-blinded challenges can be placebo-controlled (SBPC).However, in open and SBPC challenges, experimenter bias may play a role ininterpreting patient symptoms.

The typical food challenge protocol is a dose escalationstudy, usually with 15 to 30 minute dose intervals, which proceeds until aclinical effect is observed or the final dose is achieved.  The test substance,starting dose and successive incremental doses vary between protocols.  Becausereactions are assumed to be less severe at lower doses, the starting dose isgenerally in the milligram range for whole foods (Bindslev-Jensen *et al*.,2004).  Typically, food challenge studies to determine minimal eliciting dosesbegin in the low microgram range for the whole food or whole food protein.  Incrementaldoses are usually doubled or increased logarithmically, so that a reasonablenumber of incremental doses (i.e., 6 to 10) separate the starting dose from theend dose.  This final dose is usually chosen to be the normal amount in a foodserving, usually 8 to 10 gm of dried food or 60 to 100 gm of wet food (Bock *etal.*, 1988; Bindslev-Jensen *et al*., 2004).  The ability to toleratethis amount, followed by a negative open challenge on a different day, isconsidered to be evidence that the individual is not allergic to that allergen(Taylor *et**al*., 2004).

Most oral challenge studies are designed to establish adiagnosis of food allergy rather than to determine safety (Taylor *et al*.,2004).  Consequently, these studies do not start at doses below a known LOAEL.Thus, individuals who react to the starting dose are not necessarilydemonstrating a true LOAEL because it is not possible to know whether theseindividuals would have reacted to a lower starting dose without furthertesting.  A NOAEL cannot be established as long as one or more studyparticipants reacts to the starting dose.

Most elicited symptoms occur within 3 to 15 minutes after achallenge (Bindslev-Jensen *et al*., 2004).  Thus, an interval of 15minutes between challenge doses may be sufficient to confirm a negative response.Most challenge studies report the dose that elicits the first objective symptom.Because subjective symptoms may have preceded the first objective symptom atlower doses, it is often difficult to ascertain whether the reported LOAELtruly represents the lowest dose to elicit a reaction.  Subjective andobjective symptoms, including their measurement and interpretation arediscussed below.

2. Inclusion/Exclusion of Sensitive Populations

Individuals with a history of anaphylaxis to foods and infantsand children are often excluded from challenge studies for ethical reasons(Taylor *et al*., 2002).  Moreover, individuals with very high foodallergen IgE serum titers are often excluded.  Thus, food challenge studies maynot include key subpopulations of allergic individuals, who may also be themost sensitive to allergen exposure.

Individuals with allergies to a specific food have differentgenetic backgrounds and express a wide distribution of sensitivity andreactivity.  Studies have shown that there may be a range of as much asone-million-fold (106) in eliciting doses from the least sensitiveto the most sensitive individuals (Leung *et al*., 2003; Wensing *et al*.,2002b; Bindslev-Jensen *et al*., 2002).  Therefore, the inclusion orexclusion of data for highly sensitive individuals can greatly affect the NOAELdetermination for the population.  To add to this uncertainty, the mostsensitive individuals also appear to have more severe reactions (Wensing *etal*., 2002b; Perry *et al*., 2004).  The thresholds measured forpopulations that exclude these individuals may not apply to those with severeallergic disease.

3. Testing Materials 

Food challenges vary in the type of testing material used(e.g., peanut flour versus ground peanut), oral challenge vehicle (e.g., wholefood versus capsules), and in the efficacy of blinding.  Differences in thesevariables could modify the allergen distribution or concentration, affectdigestibility and absorption, influence false-positive subjective reactions,and therefore, affect interpretation of the dose-response data.

The nature of the testing material is very important, asthis can enhance or diminish the overall immunogenicity of the native allergen(Beyer *et al*., 2001; Maleki *et al*., 2003).  The matrix used(e.g., fatty substances) can delay absorption, thus affecting the time intervalto a reaction, or may affect the intrinsic allergenic properties of the food.  Also,gustatory differences in the challenge doses (because of the food matrix used)may influence subjective reactions due to poor taste or fear of consuming theallergen.  The use of capsules eliminates problems caused by taste, butbypasses the oral cavity.  Because the oral cavity plays an important role inthe initial contact and metabolism of food allergens, this may affect thesubsequent severity or character of response to the challenge dose.

4. Subjective Versus Objective Reactions

There are two types of physiologic reactions or effects thatcan occur during a food challenge - subjective symptoms, those reported by thesubject, and objective symptoms, those observed by the researcher.  Becausesubjective symptoms may be the result of non-immunological mechanisms,elicitation of objective symptoms is believed to be the more reliable indicatorof clinical reactivity to the food allergen (Taylor *et al*., 2004).

The symptoms of a severe allergic reaction are associatedwith life-threatening conditions, e.g., anaphylaxis.  However, there is noconsensus as to which of the less serious symptoms should be considered adverseeffects.  For example, can eczema be seen as a "safer" reaction thanangioedema?  Unlike well-defined toxicity endpoints, reactions to allergenicfood ingredients are part of a wide spectrum of severity that includes trivialinjury, objective systemic reactions, anaphylaxis, and death.

Subjective symptoms may be good indicators of a subsequentobjective reaction, i.e, subjective symptoms may precede or signal objectivesymptoms in a dose-dependent manner (3rd FAARP Threshold Conference,2004).  However, most challenge studies base their LOAEL determinations on thefirst objective symptom rather than a subjective symptom.  For example,although the Hourihane *et al*. (1997) study reported a threshold forpeanut proteins in the milligram range, mild subjective reactions were noted intwo individuals at doses of 100 μg of peanut protein.  Other studies donot report specific types of reactions but rather combine symptoms intocategories of mild, moderate, or severe. For example, a retrospective review of253 failed challenges at one clinic showed that the initial reaction was severein 72 (28%) and moderate in 88 (33%) of the challenges (Perry *et al*.,2004).

Currently, there is no universally accepted endpoint orresponse that can be used to predict significant harm from an allergic reaction.Anaphylaxis, a clearly significant endpoint, is a syndrome which is poorlydescribed and subject to variable interpretation.  Moreover, anaphylactic reactionsare at one extreme of a continuum of severity.  There are a number ofadditional factors (e.g., use of medicine, alcohol consumption, anxiety) thatcan significantly reduce or potentiate the impact of exposure to an allergen.  Giventhis combination of factors, a particular dose could result in mild symptomsone day and life-threatening reactions the next.

5. Anecdotal Evidence

Although a great deal of attention has been focused on theuse of challenge studies to determine threshold doses or reaction patterns forfood allergens, anecdotal reports of individuals suffering life-threateningallergic reactions from minute exposures to food allergens challenge the notionthat an allergen threshold truly exists, especially for sensitive individuals.For example, literature reports have linked kissing (Hallett *et al*.,2002; Steensma, 2003; Eriksson *et al*., 2003) and exposure to airborneparticles (Crespo *et al*., 1995; Casimir *et al*., 1997; Sackesenand Adalioglu, 2003) to allergic reactions.  Although in many of these casesthe amount of allergen exposure cannot be assessed, it is conceivable that thewhole food exposure level needed to elicit a harmful reaction is extremelylow.  In this context, it should be noted that the statistical model developedby Bindslev-Jensen *et al*. (2002) suggested that concentrations as low as700 ng for peanut and in the low microgram ranges for egg, soy flour, and cow'smilk may elicit a reaction in one in a million allergic individuals.  Althoughthis model suggests that a majority of allergic individuals would likely toleratefood allergen concentrations in the milligram range, it supports the anecdotalevidence that very low concentrations of allergen may, at some low but finiteprobability, elicit harm in highly sensitive individuals.
G. Exposure
1. Matrix Effects

Food allergens often occur as components of processed foods,and many allergic reactions occur following exposure to such allergens (Bock *etal*., 2001).  Therefore, it is important to understand how the nature orcomposition of the food (the food matrix) affects the reaction elicitationthreshold.

Very little information exists on matrix effects for themajority of allergens.  It has been reported that fat content can modify the reactionsin a peanut DBPCFC (Grimshaw *et al*., 2003).  Three of 4 subjects challengedwith peanut flour in a matrix containing 31.5% fat reacted at a higher thanexpected dose, and had reactions that were more severe than expected, based onprevious exposures to a standard recipe containing 22.9% fat.  Uponre-challenge with the 22.9% recipe, their reactions returned to expected levelswith respect to dose and severity.  The cumulative dose of peanut proteinrequired to elicit symptoms was 12 to 31 times higher when using the higher fatrecipe.  The authors suggested that the peanut allergens in the higher fatrecipe were not readily available to react with IgE on mast cells in themouth.  This was based on the observation that radioallergosorbent test (RAST)inhibition assays and enzyme linked immonosorbent assay (ELISA) detection testsshowed that peanut allergens in the higher fat mixture were less available *invitro*.  In addition, these 3 patients all had histories of an initial oralchallenge response.  The lack of an oral early warning with a high-fat food mayhave caused these patients to consume more allergen prior to the onset of othersymptoms.  By the time digestion of the fat took place in the stomach andintestine, the total dose consumed was higher, resulting in a more severereaction.

Grimshaw *et al*. (2003) further reported that theslopes of RAST-inhibition curves did not change for peanut allergens inhigh-fat versus low-fat mixtures, indicating that there was no change inantibody-binding properties.  Thus, it appears that the antigenic properties ofthe peanut flour were not altered by the higher fat matrix, and that thechanges in apparent threshold may have resulted from a combination ofphysiological and behavioral factors.

Kato *et al*. (2001) also observed a matrix effect withthe major egg allergen ovomucoid.  The ability of ovomucoid to bind IgE wasreduced in a model pasta compound composed of durum wheat and egg white.  Thisdecrease was attributed to changes in antigenicity associated with formation ofdisulfide bonds between the ovomucoid and wheat gliadins.

2. Processing Effects

Numerous studies have described alterations in allergens asa result of processing or cooking.  Various types of processing (heating,milling, fermentation, etc.) may alter the antigenic properties of allergensbecause these processes can affect the 3-dimensional structure of proteins andthus the IgE binding epitopes.  The type and extent of structural alterationsmay vary depending on the processing method.  This is especially true forconformational epitopes because they are dependant on tertiary structure (Cookeand Sampson, 1997; Vila *et al*., 2001).  For many food allergens,processing effects are inherent in the data used to characterize thresholdsbecause the test articles used in DBPCFCs are processed.  For practical reasons,the test material must be concealed in some way for the study to be "blinded."For example, the taste of peanut butter or peanut flour must be disguised inDBPCFCs for peanut allergies.  Preparation of the test material typicallyinvolves cooking or processing of the allergenic food.  In addition to alteringexisting epitopes, processing might also induce chemical or structural changesthat result in the formation of new antigenic epitopes, or neoantigens (Maleki,2004).

Altered antigenic reactivity is most commonly assessed bymeasuring changes in the binding of antibodies to extracts of raw and processedfoods. Reduced or enhanced IgE binding in such studies would suggest that thethreshold for an allergic reaction could be affected by processing.  However,definitive proof of an altered threshold requires DBPCFC testing.

The effects of processing on some major allergens haverecently been reviewed, and are discussed below (Besler *et al*., 2001;Poms and Anklam, 2004).  Variable patient responses make it difficult toconclude that a particular processing or cooking procedure affectsallergenicity in all cases.

*Peanuts*.  Extracts of roasted peanuts have been shownto bind IgE from patients at 90-fold higher levels than do similar extracts ofraw peanuts in competitive, IgE-based ELISAs (Maleki *et al*., 2000).Using immunoblot techniques, two of the major allergenic proteins in peanut,Ara h 1 and Ara h 2, were shown to be highly resistant to heat andgastrointestinal digestion following treatment in the Maillard Reaction (whichoccurs during the processing or browning of foods in the presence of heat andsugars).  Earlier studies also observed increased IgE binding and altered IgEepitopes in roasted versus raw peanuts (Nordlee *et al*., 1981).  Theallergenic proteins Ara h 1, Ara h 2, and Ara h 3 from fried or boiled peanutsbound significantly less IgE than the same proteins from roasted peanuts (Beyer*et al*., 2001), even though there were similar amounts of the allergenicproteins in peanuts processed by each method.  These studies suggest thatthresholds for boiled or fried peanuts may be higher than for roasted or rawpeanuts, at least for the three major peanut allergens.  In practical terms,the vast majority of peanuts consumed whole or in processed foods in the U.S.are roasted.  Boiled or fried peanuts are an ethnic or regional specialty andare usually eaten whole, rather than as a component of processed foods.

*Milk*. Pasteurization and homogenization did notreduce allergenicity in skin prick tests or DBPCFC (Host and Samuelsson,1988).  However, boiling milk for 10 minutes reduced IgE binding of theallergenic proteins alpha-lactoglobulin and casein by 50 to 66% and eliminatedbeta-lactoglobulin and serum albumin reactivity in skin prick tests (Besler *etal*., 2001; Norgaard *et al*., 1996).  Hypoallergenic infant formulasproduced from heat denatured or enzymatically hydrolyzed caseins or wheyproteins showed reduced allergic reactivity by immunoblot, RAST, and DBPCFC inmost milk-allergic children.  However, some severe reactions have been reported(Sampson *et al*., 1991; Saylor and Bahna, 1991).  Maillard reactionproducts in milk are reported to have increased allergenicity in skin tests(Maleki, 2004).  Allergic reactions have also been reported involving both hardand soft cheeses (Besler *et al*., 2001).

*Egg*.  Both soft and hard boiling of eggsdecreased, but not eliminate, antigen binding of rabbit antiserum to ovomucoidand ovalbumin (Besler *et al*., 2001).  Heated egg white showed a 58%decrease in IgE binding in RAST (Anet *et al*., 1985).  A decrease inpositive reactions was seen with heated egg white in 55% of egg allergicpatients using DBPCFC (Urisu *et al*., 1997).  There are reports ofallergic reaction to egg contained in cooked meatballs or hamburger (Sampson *etal*., 1992; Besler *et al*., 2001).

*Fish*. Boiling of ten species of fish failedto eliminate allergenicity in DBPCFC (Bernhisel-Bradbent *et al*., 1992).IgE binding to fish proteins in immunoblots was reduced, but not eliminated.Canning appears to reduce allergic reactions to tuna and salmon in allergicpatients tested by DBPCFC (Bernhisel-Broadbent *et al*., 1992).  IgEbinding of allergenic proteins from canned fish was reduced by 98 to 99% comparedto boiled fish.  IgE binding studies indicate that fish allergens are presentin surimi.

*Shellfish*. Boiling does not reduce reactivity ofshrimp allergens (Daul *et al*., 1988; Naqpal *et al*., 1989).

*Soy*. Heating soybeans at 100�C for 60 minutes doesnot completely eliminate IgE binding to allergenic soy proteins (Burks *et al*.,1992).  Various soybean products including sprouts, soy sauce, hydrolyzed soyprotein tofu, miso, and lecithin all retained IgE-binding activity (Besler *etal*., 2001).  IgE binding proteins have been found in soy lecithin (Gu *etal*., 2001; Porras *et al*., 1985; Paschke *et a*l., 2001).  Allergicreactions to soy lecithin have also been reported (Renaud, 1996; Palm, 1999).The protein content of soy lecithin has been reported to vary between 2.8-202mg per 100 g (Besler *et al*., 2001; Paschke *et al*., 2001).  IgEbinding proteins have been detected in unrefined soybean oils (Paschke *et.al*., 2001), but inconsistently in refined oil (Awazuhara *et al*.,1998; Paschke *et al*., Errahali *et al*., 2002)

*Tree nuts*.  Protein extracts of severalhazelnut-containing products demonstrated less IgE binding than raw hazelnutextracts, suggesting that processing reduced allergenicity.  However, some IgEbinding capacity remained (Wigotzki *et al*., 2001).  Several cases ofanaphylaxis have been described for other processed nut-containing products,suggesting that processed tree nuts in general retain allergenic activity(Besler *et al*., 2001).  Roasting, blanching, autoclaving, or microwavingdid not change the ability of animal antisera to bind almond proteins(Venkatachalam *et al*., 2002).

3. Detecting and Measuring Allergens

There are several factors that make it difficult to detectand measure food allergens.  These include sampling problems and difficultiesin quantifying proteins, particularly allergenic proteins, in a wide variety offoods.  Further, an allergen may be a minor component of a highly complex,heterogeneous food.  The food matrix can sequester allergens, hinderingdetection, while not significantly affecting allergenicity.  It is alsodifficult to estimate the amount of a food allergen that may be present fromthe result of an assay that only measures protein, particularly when there ismore than one allergenic protein.

The only commercial methods that have been shown to detectfood allergens reliably use immunological techniques such as ELISA (Poms *etal*., 2004; Krska *et al*., 2003; Popping *et al*., 2004).  Inmany cases, these methods were designed to detect representative biomarkers,not necessarily a specific allergenic protein.  Many kits contain polyclonalantibodies that detect both non-allergenic and allergenic proteins.  Forexample, the peanut ELISA assays that have completed Multiple LaboratoryPerformance Tested validation are designed to detect multiple proteinsindicative of the presence of the food (e.g., peanuts), not to detect orquantify specific allergenic proteins (Park *et al*., 2005).  There are novalidated detection methods or commercially available kits for most food allergensor for specific allergenic proteins.

The FDA and AOAC investigated the ability of threecommercial peanut test kits [BioKits Peanut Testing Kit (Tepnel), Veratox forPeanut Allergens (Neogen Corp.), and RiDASCREEN Peanut (R-Biopharm GmbH)] tomeasure accurately peanuts in four food matrices (cookies, ice cream, milkchocolate, and breakfast cereal) (Park *et al*., 2005).  The validationstudy, requiring 60 analyses of test samples at the target level of 5�gpeanut/g of food and 60 analyses of  "peanut-free" controls, was designed toensure that the lower 95% confidence limit on the true sensitivity andspecificity rates exceeded 90% (Park *et al*., 2005).  The results fromthis study showed that all the test kits correctly allocated the test samples atthe target level.  No comparable studies have been completed for any other foodallergen.

Scientific practice is to calibrate, standardize, andvalidate assays and commercial test kits for each food product because minordifferences in the matrix change the recovery and detection of specific foodproteins.  Standardization requires the preparation of samples identical to thetest sample and containing known amounts of a specific food allergen.  Nevertheless,because different antibody-based assays recognize different protein epitopes, variableresults may be obtained using different test systems.  This variability wasevident in results obtained in the Food Analysis Performance Assessment Scheme(FAPAS®) supervised proficiency studies of wheat, peanut, egg, and milk testkits (FAPAS Reports 2705 and 2705 Supplement (wheat), 2708 (peanuts), 2710 (eggand milk).

Highly variable food matrices and the nature of foodproduction also create sampling challenges.  The distribution of allergenicproteins within whole foods is not necessarily homogenous, and allergenicingredients may not be evenly distributed throughout processed foods.  Inaddition, cross-contact may result in a heterogeneous distribution of allergenswithin or on a food.  For example, nuts may be introduced into chocolate on aproduction line where nut-containing and nut-free products are processedsequentially.  In this case, cross-contact is most likely to occur at thebeginning of a production run for the nut-free product.  Thus, allergen testingusing chocolate taken from the end of a production run might not adequatelycharacterize the risk.

For a food product, development of a scientifically sound samplingplan that includes a statistical analysis of the probability that all allergensare detected, ensures that any allergens present are accurately measured.Important sampling questions that need to be considered include whether theallergen is likely to be heterogeneously distributed within the batch; thenumber of samples per batch that should be tested; which batches should betested; which portion of a run should be tested; and how to obtain a specificdegree of confidence (e.g., 95% confidence) that no allergen is present.

The currently available commercial assays are designed forthe detection of food allergens, not specific allergenic proteins.  Tests forspecific allergenic proteins (e.g., Ara h 1 in peanut) may provide usefulsupplemental information, but these tests are research tools and are notcurrently viewed as practical for routine use.
H. Collective Allergens
Three of the major food allergens identified in the FALCPAare actually groups of foods: crustaceans, fish, and tree nuts.  It is possiblethat proteins from two or more species within each of these "collectiveallergens" might be present in a food and the available analytical methods areunable to distinguish between species in a group.  Therefore, it may benecessary to consider total protein levels from all species in a group ratherthan the level of protein from each species. In addition, an individualallergic to one species is likely to also be allergic to other species in thegroup.

The ability of available test methods to distinguishdifferent species within each group of "collective allergens" varies.  To date,there are no commercially available test kits for finfish proteins and only onefor crustacean tropomyosin.  Ben Rejeb *et al*. (2003) reported thedevelopment of an ELISA for shrimp that showed significant cross-reactivitywith other crustaceans.  There are three commercially available tree nut testkits (two for hazel nut, one for almond), but the species specificity of thesekits is not clear.  Hlywka *et al*. (2000) showed that an almond ELISAdetected protein from seven other tree nuts.  The hazel nut ELISA developed byHolzhauser *et al*. (2002) showed cross-reactivity with other nuts, andthe walnut assay developed by Niemann and Hefle (2003) reacted with three othernut species.  Wei *et al*. (2003) developed an ELISA for cashew thatshowed cross-reactivity with several other nuts. Ben Rejeb *et al*. (2003)developed a hazel nut-specific ELISA that did not cross-react with other nuts,and Clemente *et al*. (2004) developed a Brazil nut assay with"negligible" cross reactivity to five other nut species.

Although not likely to be useful for routine screening ortesting, techniques such as LC/MS are being used to identify specificallergenic proteins in complex food matrices (Shefcheck and Musser, 2004).These approaches may be useful either as confirmatory tests or for characterizationof foods containing several allergens.

*Crustacean Shellfish*.  Allergenic cross-reactivityamong crustaceans is considered to be common.  Sicherer (2001) estimated thatthere is a 75% probability that a shrimp-allergic individual will also react toat least one other crustacean.  Waring *et al*. (1985) reported that 11 of12 (92%) patients with skin prick reactions to shrimp also had positive skinprick reactions to at least one other crustacean.  Similarly, Daul *et al*.(1987) showed that between 73 and 82% of shrimp allergic patients had positiveskin prick tests to another crustacean.  Chiou *et al*. (2003) showed thatsera from 20 of 32 individuals with either shrimp- or crab-reactive IgE werereactive to both species.  Further, inhibition studies with 15 of thesecross-reactive sera showed relatively high affinity for both allergens.  Thebasis for this high rate of cross-reactivity appears to be sensitivity to thehighly conserved protein tropomyosin, which is considered to be a panallergen(Daul *et al*., 1993; Leung *et al*., 1999; Sicherer, 2001).

*Fish*.  Allergenic cross-reactivity among fish specieshas been described in the clinical literature, but appears to be less commonthan among species of crustacea.  Both Sicherer (2001) and Sampson (1999) estimatethat there is a 50% probability that an individual allergic to one fish specieswill also react to at least one other fish species.  Helbling *et al*.(1999) reported that 4 of 14 (29%) fish allergic patients reacted to two ormore species in DBPCFC tests.  Bernhisel-Broadbent *et al*. (1992)reported that 3 of 10 (30%) fish allergic patients responded to more than onefish species in oral challenges, but that skin prick tests were positive tomultiple species for all of these patients.  Similarly, Hansen *et al*.(1997) showed that eight cod allergic patients all had positive skin pricktests with two other fish species.  The data presented in Pascual *et al*.(1992) suggest that at least 80% of a group of 79 fish allergic children hadIgE antibodies to two or more fish species.  In some cases, cross-reactivityhas been shown to reflect the presence of one of more closely relatedallergenic proteins in different species (Pascual, 1992; Hansen *et al*.,1997; Leung *et al*., 1999; Hamada *et al*., 2003).

*Tree Nuts*.  The prevalence of cross-reactivity amongtree nuts is difficult to determine accurately for several reasons:  the highproportion of severe reactions among nut-allergic patients makes it dangerousto carry out oral challenge studies, many published works test for reactivityto a small number (and variable assortment) of tree nuts, and studies oftencombine tests for tree nuts and peanuts.  Nevertheless, Sicherer (2001)estimates that a tree nut allergic patient has a 37% chance of being allergicto two or more species of tree nut, and Sampson (1999) estimates that theprobability of multiple tree nut sensitivities at greater than 50%.  Ewan(1996) reported that 12 of 22 (55%) of tree nut allergic patients responded tomultiple tree nuts by skin prick tests.  Sicherer *et al*. (1998) andPumphrey *et al*. (1999) both used *in vitro* IgE testing and foundmultiple sensitivities in 37% and 61% of tree nut allergic patients,respectively.  There are a number of studies that report cross-reactions in oneor a few patients (e.g., Teuber and Peterson, 1999; Ibanez *et al*., 2003;de Leon *et al*., 2003; Asero *et al*., 2004).  The complex patternof cross-reactivity among the tree nuts may reflect the fact that severaldifferent panallergens (lipid transfer proteins, profilins, Bet v1-relatedproteins) and evolutionarily conserved proteins (seed storage proteins) occurin various tree nuts (Roux *et al*., 2003).
I. Published Challenge Studies
An extensive literature review was conducted from November2004 through April 2005 that included key word, author, and "related article"searches of the PubMed database and analysis of citations found in thepublished literature. Sixteen publications with quantitative dose-response datafrom DBPCFC testing were reviewed to identify those that contained data thatcould be used to estimate LOAEL levels for the major food allergens.  Thesestudies are described in more detail in Appendix 2.  Thirteen (80%) of thesereport results from testing adults; the remaining three tested infants and children.In four cases, the population being studied was not specifically chosen to befood allergic, and a large fraction of the individuals in these populations didnot respond to the highest doses tested.  In seven studies (44%), patientsreacted to the lowest dose tested, and in three studies there was insufficientinformation to determine either the lowest dose used or the number of patientswho responded to that dose.  The most sensitive population was seen byHourihane *et al*. (1997b), who reported that 67% of the patients testedreacted to "peanut rubbed on the lip," including one severe reaction.

*Peanut*.  Hourihane *et al*. (1997) observed thelowest measured dose of an allergen that provoked a reaction (i.e., a LOAEL), 0.1mg of peanut protein provoked subjective reactions in two patients and 2 mg ofpeanut protein provoked an objective reaction in one patient.  Objectivereactions were observed in two other patients on exposure to 5 mg of peanutprotein.  Wensing *et al*. (2002a) also reported a LOAEL of 0.1mg forsubjective reactions in two of 26 peanut allergic individuals tested. The LOAELfor the intial objective symtom was 10 mg.  Several other papers reportedLOAELs of 25-100 mg of peanut protein for objective reactions (May, 1976;Hourihane *et al*., 1997; Bock *et al*., 1978).

*Egg*. A wide range of LOAELs have been observed foregg.  Caffarelli *et al.* (1995) reported a LOAEL of 0.5 mg of dried wholeegg (approximately 0.45 mg protein).  Bock *et al*. (1978) reportedobserving an objective reaction with 25 mg of whole egg (approximately 1 mgprotein), although the data are difficult to interpret as presented.  Incontrast, Eggesbo *et al*. (2001) report a LOAEL of 1 g of whole egg(approximately 250 mg of protein) for an objective reaction.

*Milk*. Relatively consistent LOAELs have beenreported for milk.  Bellioni-Businco *et al*. (1999) found a LOAEL of 1 mlof whole milk (approximately 350 mg of protein) with children, and Pastorello *etal*. (1989) found a LOAEL of 0.5 g of freeze-dried milk (approximately 185mg of protein) with adults.

*Soy*. LOAELs of approximately 50 and 88 mg proteinhave been reported for soy (Zeiger *et al*., 1999; Magnolfi *et al*.,1996).

*Tree Nut*. Hazel nut is the most commonly studiedtree nut.  Wensing *et al*. (2002b) observed reactions to 1 mg of hazelnut protein in 4 of 29 patients, which was the lowest dose tested.  Hansen *etal*. (2003) found a LOAEL of approximately 30 mg of hazel nut protein,although it is not clear whether this was the lowest dose tested.

*Fish*. Hebling *et al*. (1999) reported a LOAEL of 50 mg for catfish protein.
J. Food Treatments to Reduce Allergenicity
The best example of food products that are processed torender them less allergenic are hydrolyzed infant formulas.  Enzymatichydrolysis of cow's milk protein or derivatives (i.e., casein, whey) has beenshown to significantly reduce the levels of both total and allergenic (e.g., b-lactoglobulin) protein (Host and Halken,2004).  The degree of protein reduction depends on the method of hydrolysis.There is ample clinical evidence to suggest that hydrolyzed formulas havereduced allergenicity in comparison to intact milk formulas (Amer. Acad. Ped.,2000; Host and Halken, 2004).  Furthermore, there is evidence that the use ofcertain hydrolyzed formulas may also delay or prevent the development of cow'smilk allergy (CMA) in high-risk infants (Host and Halken, 2004).

Hydrolyzed formulas contain varying amounts of residualprotein, including allergenic proteins, which can be detected using either *invitro* or *in vivo* methods (Giampietro *et al*., 2001; Docena *etal.*, 2002).  Both extensively and partially-hydrolyzed formulas can causeallergic reactions, including anaphylaxis, in sensitive infants (Saylor and Bahna,1991; Schwartz and Amonette, 1991; Tarim *et al.*, 1994; Ammar *et al.*,1999; Giampietro *et al.*, 2001; Host and Halken, 2004).  Thus, theresidual milk proteins (or peptides resulting from their partial hydrolysis) inthese formulas still retain immunologic activity.  In general, the higher thelevel of residual protein, the higher the risk for a reaction.  Althoughpartially hydrolyzed formulas tend to show higher residual protein levels, thedegree of hydrolysis cannot always be used as a predictor of the degree ofallergenicity.  Hydrolysis methods are not standardized, and formulasundergoing similar treatments may vary considerably in their residual proteinlevels.  Additional processing, such as heat treatment and ultrafiltration, mayfurther reduce residual protein levels in certain products (Host and Halken,2004).

In 1989, the American Academy of Pediatrics (AAP) determinedthat a formula could be considered "hypoallergenic" if challenge studiesshowed, at a minimum, 95% confidence that 90% of allergic infants would notreact adversely to the formula (Amer. Acad. Ped., 1989).  Since this time, anumber of DBPCFC studies using various infant formula preparations have beenperformed in infants with CMA (Sampson *et al.*, 1991; Sampson *et al.*,1992; Giampietro *et al.*, 2001; Sicherer *et al.*, 2001), and asubstantial number of infant formulas have met this criterion forhypoallergenicity.  Even though they note that extensively hydrolyzedhypoallergenic formulas contain residual proteins and may provoke allergicreactions in infants with CMA, the AAP currently recommends these formulas asalternatives for infants with CMA stating that at least 90% of these infantswill tolerate the formula (Amer. Acad. Ped., 2000).

Newer technologies, such as genetic modification, are beingdeveloped to reduce allergenicity by removing, silencing, or modifying thegenes for specific allergenic proteins within foods (Tada *et al*., 1996;Herman *et al.*, 2003; Dodo *et al.*, 2005; Gilissen, 2005).  Todate, however, there is no example of a food allergen that has been renderedcompletely devoid of allergenic activity using these methods.  This is due tothe fact that each food contains a number of allergenic proteins, each withmultiple allergenic epitopes.  Unless these methods can eliminate all of theseproteins, or modify all allergenic epitopes, the remaining proteins or epitopescould still elicit a reaction in sensitive individuals.
III. Celiac DiseaseA. Introduction
Celiac disease (also known as celiac sprue) is a chronicinflammatory disorder characterized by mucosal damage of the small intestineleading to gastrointestinal illness, nutrient malabsorption, and a wide rangeof clinical manifestations.  There is a consensus opinion that celiac diseaseis caused by an aberrant (T lymphocyte) immune response to dietary glutenspredominantly found in wheat, barley, and rye (NIH, 2004).  However, there isevidence that at least some persons who have celiac disease also cannottolerate oats (Lundin *et al*., 2003; Arentz-Hansen *et al*., 2004).Those individuals who have a genetic predisposition to celiac disease react topeptides within the proline- and glutamine-rich protein fractions of thegrains.  For affected individuals, celiac disease is a lifelong condition and,if not treated, is associated with significant morbidity and increasedmortality (Fasano, 2003; Corrao *et al*., 2001; Dewar *et al*., 2004).There is no cure for celiac disease.  Strict avoidance of potentially harmfulconcentrations of glutens in the diet is the only known means of completelypreventing the clinical and pathological complications of celiac disease.
B. Mechanism of Pathogenesis
Celiac disease is characterized by injury to the mucosa ofthe small intestine and specifically targets the fingerlike projections, calledvilli, where absorption of key nutrients takes place (Figure III-1).  Thisinjury is believed to be due to an autoimmune disorder involving modificationof the antigenic presentation of gluten in the intestinal tract of geneticallypredisposed individuals expressing the major histocompatibility haplotypesHLA-DQ2 or HLA-DQ8 (Farrell and Kelly, 2002; Fasano, 2003).  In theseindividuals, binding of the enzyme tissue transglutaminase (tTG) to wheat gluten(a glutamine rich protein) potentiates uptake and presentation by antigen-presentingcells in the lamina propria, triggering a vigorous T-cell response (Schuppanand Hahn, 2002), leading to  production of IgG and IgA directed to wheat glutenpeptides (i.e., gliadins and glutenins) and IgA to tissue transglutaminase(tTG).  The activated T-cells are responsible for the self-perpetuating mucosaldamage seen in celiac disease (Fasano and Catrassi, 2001).  Thisimmune-mediated damage occurs in two compartments, the epithelium and thelamina propria (Green and Jabri, 2003).  Early intestinal disease ischaracterized by an increased number of intestinal intraepithelial lymphocytes(IELs).  As the disease progresses, increasing numbers of lymphocytes andplasma cells infiltrate the lamina propria.  This increase in the numbers of cellsleads to elongation of intestinal crypts and shortening of villi, whicheventually results in partial or total villous atrophy (James, 2005).  Eliminationof intestinal gluten results in modification of T lymphocyte and antibodyresponses and, in most cases, full mucosal recovery (Kaukinen *et al*.,1999; Fasano and Catassi, 2001).

**Figure III-1 Mechanism of Celiac Disease**
C. Range of Adverse Effects
The clinical manifestations of celiac disease are highlyvariable in character and severity and depend largely on the age andimmunological status of the individual, the amount, duration, or timing ofexposure to gluten, and the specific area and extent of the gastrointestinaltract involved by disease (Dewar *et al*., 2004).** These clinicalmanifestations can be divided into gastrointestinal, or "classic," andnon-gastrointestinal manifestations.  Gastrointestinal manifestations usuallypresent in children 4 to 24 months old and include acute symptoms of abdominalpain and cramping, bloating, recurrent or chronic diarrhea in association withweight loss, poor growth and symptoms consistent with nutrient deficiency, and(in rare cases) a life-threatening metabolic emergency termed celiac crisis,characterized by hypokalemia and acidosis secondary to profuse diarrhea(Farrell and Kelly, 2002; Baranwal *et al*., 2003).  Non-gastrointestinalmanifestations are more insidious and highly variable and are the commonpresenting symptoms in older children and adults.  These manifestations are frequentlythe result of long-term nutrient malabsorption, including iron deficiencyanemia, short stature, delayed puberty, infertility, and osteoporosis orosteopenia (Fasano, 2003).  In children, progressive malabsorption of nutrientsmay lead to growth, developmental, or neurological delays (Catassi and Fasano,2004).  Extra-intestinal manifestations such as dermatitis herpetiformis,hepatitis, peripheral neuropathy, ataxia, and epilepsy have also beenassociated with celiac disease.  Individuals with untreated celiac disease areat increased risk for potentially serious medical conditions, such as otherautoimmune diseases (e.g., Type I diabetes mellitus) and cancers associatedwith high mortality (Farrell and Kelly, 2002; Peter**s***et al*.,2003; Catassi *et al*., 2002).  For example, individuals with celiacdisease have an 80-fold greater risk of developing adenocarcinoma of the smallintestine and a greater than two-fold increased risk for intestinal orextraintestinal lymphomas (Green and Jabri, 2003).  The latter complicationsare responsible for nearly two thirds of deaths due to celiac disease and are amajor reason for the nearly two-fold increase in overall mortality of adultpatients with celiac disease compared to the general population (Corrao *et al*.,2001).

Currently, individuals with clinical manifestations, or"symptomatic" celiac disease, are believed to represent a small portion of thetotal celiac population.  A much larger number of individuals have "silent"celiac disease, characterized by positive serology and limited involvement ofthe GI tract.  There is an even larger population with "latent" celiac disease,individuals who are positive for serological markers or genetic susceptibilityto disease but show no intestinal mucosal involvement.  Individuals in theselatter two categories may have atypical disease manifestations or, in mostcases, be entirely asymptomatic.  It is generally accepted that individualswith silent or latent disease have aberrant immune responses following exposureto dietary glutens and are, therefore, at increased risk for both acute andlong-term complications of celiac disease (Fasano, 2003; Schuppan, 2000).  Thelong-term benefit of strict gluten avoidance for these individuals is unproven(Green and Jabri, 2003).
D. Prevalence
Until recently, celiac disease was considered to be a raredisorder in the U.S., with an estimated prevalence rate of 1:10,000 (Talley,1994).  However, a large epidemiological study screened more than 13,000 peoplein 23 states and estimated a prevalence rate of 1:133 within the general U.S.population (Fasano, 2003).  The National Institutes of Health ConsensusDevelopment Conference Statement on Celiac Disease currently estimates that 3million Americans, a little less than 1 percent of the population, may haveceliac disease (NIH, 2004).  Celiac disease occurs widely among North Americanand European populations, where wheat is a staple food, but is infrequent amongnative descendents of China and Japan and those with an African-Caribbeanbackground, where wheat is not as widely consumed (Farrell and Kelly, 2002).

Precise prevalence data for celiac disease are notavailable.  This disease is often misdiagnosed as another gastrointestinalmalabsorptive disorder (e.g., Crohn's disease, irritable bowel syndrome) due tosimilarities in their symptoms.  Due to the existence of silent or latent cases,it is assumed that the incidence of celiac disease is underreported.  Theseforms of celiac disease may go undetected in individuals for years before theydevelop symptoms causing them to seek medical attention (Green and Jabri,2003).  M�ki and Collin (1997) postulated that there are many more currentlyhealthy individuals who are genetically predisposed to developing celiacdisease in future years than there are individuals who are now affected byceliac disease.  Only recently has the medical community become more aware ofthe need to screen for celiac disease when patients experience health problemsthat may be associated with the disease or when patients have family members,especially first- and second-degree relatives, who have celiac disease (NIH,2004).
E. Celiac Foods of Concern
Celiac disease is caused by an immune response ingenetically predisposed individuals to specific storage proteins, commonlyreferred to as "glutens," that occur naturally in cereal grains (Shan *et al*.,2002).  Technically, "gluten" isa term applied *specifically* to the combination of the prolaminproteins called "gliadins" and the glutelin proteins called "glutenins" foundin wheat.  However, the term "gluten" has been used generically to referto prolamin and glutelin protein mixtures found in other cereal grains (Kasarda,2005, personal communication).  Although all cereal grains containprolamin and glutelin proteins, these proteins are not identical in differentgrains.  These proteins differ in their amino acid sequences in differentgrains, and not all have been shown to evoke an abnormal immune response thataffects the intestinal lining of persons genetically susceptible to celiacdisease (Kasarda, 2003).  The term "gluten" will be used in this report in themore general sense of the combination of both prolamin and glutelin proteinsfound in cereal grains.  

The grains considered to be capable of  producing adverseeffects in individuals with celiac disease include the different species ofwheat (e.g., durum, spelt, kamut), barley, rye, and their cross-bred hybrids(e.g., triticale, which is a cross between wheat and rye) (Kasarda, 1994; Kasarda,2004).  There is also evidence that some individuals with celiac disease mayreact adversely to oats (Lundin *et al.*, 2003; Arentz-Hansen, 2004).  Thesegrains are all members of the grass family (*Gramineae,* also known as *Poaceae)*and are closely related taxonomically.  The  cereal grains assumed to be safefor persons with celiac disease include amaranth, buckwheat, corn, Indian ricegrass, Job's tears, millet, quinoa, ragi, rice, sorghum, and teff (or tef) (Kasarda,2001; Kasarda, 2004b; Kupper, 2004).

The grain prolamins of concern include gliadin in wheat, secalinin rye, hordein in barley (Thompson, 2001; Green and Jabri, 2003; Kagnoff, 2005)and possibly avenin in oats (Arentz-Hansen, *et al*. 2004; Lundin, *etal.*, 2003).  There is substantial evidence that both prolamin proteins(i.e., gliadins) and glutelin proteins (i.e., glutenins) in wheat affectindividuals with celiac disease (Shan *et al.,* 2002; Hausch *et al*.,2002; Vader*et al*., 2002; van de Wal *et al*., 1999; Molberg *et al*.,2003).

Wheat gliadin subtypes alpha, gamma, and omega, have been shownto affect individuals with celiac disease (EFSA, 2004).  Rye, barley andtriticale are also considered to affect individuals with celiac disease becausethey are taxonomically related to wheat, express peptides structurally similarto those found in wheat, and have been shown to affect individuals with celiacdisease (Vader *et al.,* 2002; Kasarda, 2001; Kasarda, 2004b).  In contrast,the prolamins in other cereal grains (e.g., zein in corn and orzenin in rice) havebeen shown not to affect individuals with celiac disease (EFSA, 2004; Kasarda,2004b). However, much is still unknown about which proteins in the differentgrains can affect individuals with celiac disease (Kasarda, 2001).

Analytical information is not available on the actual amountof gluten proteins in different grain-derived food ingredients or finishedfoods.  For single ingredient foods made from wheat, rye, barley, triticale, andoats, the simple presence of "protein" in that food may be used as an indicatorthat gluten proteins are present.  The USDA *National Nutrient Database forStandard Reference, Release 17* (USDA, 2004), the major source ofcomposition data for foods in the U.S., includes hundreds of food items thatcontain wheat, rye, barley, triticale or oats as an ingredient.  Wheat, inparticular, is used to manufacture a wide range of food ingredients andfinished foods.  Rye, barley, triticale, and oats are used to make substantiallyfewer food products.

Koehler and FDA (2005) estimated the average amount of totalgrain and individual types of grain available for consumption per person in the  U.S., and the total exposure to gluten-forming proteins that would resultfrom this grain consumption.  The estimated mean daily consumption rate was approximately250 grams of grain per capita.  Wheat provided 180 of the 187 grams per personper day of grains that are of concern for individuals with celiac disease.

There is no consensus as to whether oats present a hazardfor all individuals with celiac disease.  Several studies, including one that lasted5 years, have reported that most celiac study participants both preferred andtolerated moderate amounts (e.g., 50-70 grams daily) of oats (Janatuinen *etal*., 1995; Janatuinen *et al*., 2000; Janatuinen *et al*.,2002).  However, two smaller, more recent studies that investigated the effectsof daily consumption of 50 grams of oats by individuals with celiac disease suggestthat oat proteins can elicit symptoms in some sensitive celiacs (Arentz-Hansen *etal*., 2004; Lundin *et al*., 2003).  The oats used in these studieswere tested to ensure that they did not contain any gluten proteins from wheat,rye, or barley.
F. Gluten Contamination of Grains
In the U.S., most commercially available oat products arebelieved to contain some gluten proteins from wheat, rye, or barley due tocross-contact with these grains during growth, harvest, transport, storage, or processing(Kasarda, 2001; Kasarda, 2005 personal communication; AGA, 2001; Thompson,2003).  In a recent study, Thompson (2004) analyzed four lots of three brands ofrolled or steel-cut oats commercially available in the U.S. for prolamins fromwheat, barley, or rye.  For one brand, all samples contained 338 to 1807 ppmgliadin (expressed as �g per mgof food product).  For each of the other two brands, the level of gliadindetected in all but one lot ranged from 12-725 ppm in one brand and 120-131 ppmin the other brand.  Thus, only one lot of these two brands was negative forgliadin.  Thompson (2004) concluded that none of these three brands could beconsidered a reliable source of oats free of potentially harmful glutenproteins.

Grains that do not contain gluten can become contaminatedwith grains that contain gluten at any step in the farm-to-table continuum, particularlyif shared equipment is not thoroughly cleaned between uses.  It is difficult,if not impossible, to prevent all cross-contact situations, considering thetons of grain handled by farm equipment, bulk storage, and transport containerson a daily basis.  In fact, the Official United States Standards for Grains(USDA, 1999) assume that most grains that have an established U.S. standardwill contain a small percentage of other grains.
G. Gluten Challenge Studies
There is little information in the literature on minimaldisease-eliciting doses of gluten for sensitive individuals.  Gluten challengeshave generally been performed in individuals where diagnosis is uncertain (e.g.,infants, Laurin *et al*., 2002) or in individuals with unclear intestinalpathology results (Wahab *et al*., 2001).  Challenges have also beenperformed to determine the time of disease relapse after a prolonged period ofgluten avoidance (Mayer *et al*., 1989).  In most cases, gluten challengeshave been performed to elicit or confirm disease rather than to measuresensitivity (Farrell and Kelly, 2002).

There is no standard protocol for gluten challenges, andchallenge studies have varied greatly in amount and duration of gluten exposure.Although some studies have been designed to determine the acute effects (i.e.,after 4 hours) of exposure to gluten (Sturgess *et al*., 1994; Ciclitira *etal*., 1984), most challenges consist of an open challenge to a fixed orincremental dose of daily gluten over a minimum period of 4 weeks.  Manychallenge studies use a high exposure (> 10 gm/daily) to gluten,because this is believed to shorten time to disease confirmation or relapseand, therefore, to minimize discomfort to subjects (Rolles and McNeish, 1976).However, some studies have shown that low daily exposures to gluten can elicita disease response (Catassi *et al*., 1993; Laurin *et al*., 2002;Hamilton and McNeill, 1972).

Catassi *et al*. (1993) reported that children, whoseceliac disease had previously been controlled on gluten-free diet, had evidenceof intestinal mucosal or immunological changes (changes in intraepitheliallymphocyte counts and the villous height to cryptdepth ratio) following 100 mg or 500 mg of daily gliadin over 4 weeks;this corresponds to 200 mg and 1000 mg of daily gluten respectively (Collin *etal*., 2004).  The degree of inflammation was dose-dependent.  However, thisstudy had several important limitations, which include the short term follow up(4 weeks), testing in young children, the small number of subjects (n=20), andthe lack of control groups.  In addition, although gliadin is believed to be themajor immunogenic portion of gluten, T cells from the small intestine of CDpatients have been shown to be responsive to peptides from the glutenin portionas well (Van de Wal *et al*., 1999).  Thus, the Castissi *et al*.(1993) study was also limited by the use of gliadin rather than gluten.  Estimatingpotential harm by extrapolating from gliadin levels may not be representativeof the harm from total gluten exposure.

A studycurrently in progress [The Italian Microchallenge Study] has extended the scopeof these earlier findings by evaluating the effects of exposure to either 10 or50 mg of purified gluten per day for 3 months with a population of 36 celiacdisease individuals in a double-blind, placebo-controlled study (Catassi *etal*., 2005).  Preliminary unpublished results suggest that minimal mucosalabnormalities occur with a strict gluten-free diet, that both 10 mg and 50 mgdaily gluten are well-tolerated, but that there is a trend for mucosal changesto occur at the 50 mg dose.  These results can be compared to estimated glutenexposures from gluten-free diets containing various levels of glutencontamination (Table III-1, from Collin *et al*., 2004, reproducedbelow).  Fasano (2005 personal communication) used these values to suggest thata conservative threshold for gluten exposure for sensitive individuals wouldlie between 20 and 100 ppm.
Table III-1.  Estimated Daily GlutenConsumption from Combinations of Different Amounts of Food Containing DifferentLevels of Gluten  Gluten Content  in Food (ppm)    Daily Amount of Gluten-Free  Food Consumed (g)    50    100    200    300        ----------------Daily Amount of Gluten Consumed (mg)-----------------    200    10    20    40    60    100    5    10    20    30    50    2.5    5    10    15    20    1    2    4    6  Source: Collin *et al*.,2004. Note:  Gluten content in foodmultiplied by food consumed equals gluten consumed.	 Six slices of bread isequivalent to approximately 100 g baking mix. ppm=mg/kg
In an alternate approach,Collin *et al*. (2004) analyzed gluten levels in a number of differenttypes of wheat starch (n=24) and naturally gluten-free (n=59) flours consumedby 76 individuals with celiac disease who had been on gluten-free diets for 1to 10 years. These individuals had no reported evidence of mucosaldeterioration or significant provocation of symptoms while on this diet. Therange of gluten found in these products was 0 to 200 ppm.  Collin *et al*.(2004) then estimated that the total daily flour consumption for theseindividuals to be 10-300 gm (median 80 gm).  Based on this estimate and thegluten content of the flour, a chart depicting estimated daily gluten exposureswas devised (Collin *et al*., 2004).   Collin *et al*. (2004) usedthis chart and data from low dose gluten challenge studies to suggest the useof a threshold of 100 ppm gluten.  The main limitations of this study includelack of a prospective study design (for actual dose-response information) andthe lack of information detailing diagnostic assessment (i.e., minimal mucosalinvolvement) for characterizing mucosal relapse in these individuals.
H. Measuring Gluten in Food
Currently, commercial immunology-based ELISA test kits forthe detection of gluten in foods are manufactured by Immunotech (CzechRepublic), Ingenasa (Spain), Morinaga (Japan), Diffchamb (Sweden), NeogenCorporation (U.S.), R-Biopharm (Germany), and Tepnel BioSystems (U.K.).  All ofthese detect prolamins, the proteins found in soluble aqueous-alcohol extractsfrom cereals.  None is designed to detect all proteins associated with celiacdisease.  Five of the assays have separately undergone multi-laboratoryvalidation studies (Skerritt and Hill, 1991; Akiyama *et al*., 2004; Gabrovsk�*et al*., 2004; Immer *et al*., 2003).  Each of these studiesemployed different target levels and matrices.  The Tepnel kit was validated byAOAC at 100 ppm (Skerritt and Hill, 1991).  All the ELISA kits rely on thepreparation of an aqueous-alcohol extracts as analytical samples, and four ofthe manufacturers include the use of reducing-denaturing conditions for theanalysis of baked goods.  During the 25th session of the CodexCommittee on Nutrition and Foods for Special Dietary Uses in 2003, the R5-MendezELISA method, which entails the use of reducing - denaturing conditions, wasforwarded to the Codex Committee on Methods of Analysis and Sampling for endorsement(Codex Alimentarius Commision, 2003).  These ELISA test kits cross react, todiffering degrees, with prolamins derived from wheat, rye, and barley.  None ofthe test kits cross-reacts with protein extracts from oats (Gabrovsk� *et al*.,2004; Nonaka, 2004; Abouzied, 2004; Brewer *etal*., 2004).  As such, the ELISA test kits do not provide protection toindividuals with celiac disease who are sensitive to oats (Peraaho *et al*.,2004; Storsrud *et al*., 2003; Arentz-Hansen *et al*., 2004; Lundin *etal*., 2003).  Proficiency testing studies conducted by the Food AnalysisPerformance Assessment Scheme (FAPAS®) have shown variability between theprolamin ELISA test kits (FAPAS Series 27 Round 05, Report No. 2705, 2003),indicating that further validation studies for these kits need to be carriedout under comparable conditions.  In addition to ELISA test kits, two of themanufactures, Tepnel BioSystems and R-Biopharm, market lateral flow devices forthe detection of gluten.  To date, neither of these have been validated.

At this time there is no correlative information on theefficacy of using these tests to predict or help prevent adverse effects in individualswith celiac disease.
I. Gluten-Free Labeling
Although gluten-free diets are considered the only effectivetreatment for individuals with celiac disease, it has been recognized that itis difficult, if not impossible, to maintain a diet that is completely devoidof gluten (Collin *et al*., 2004).  Therefore, several attempts have beenmade to define gluten-free in regulatory contexts.  Efforts by the CodexAlimentarious to define a standard for gluten-free date back to 1981.  At thattime, due to the lack of sensitive, specific analytical methods, a thresholdvalue of 0.05 g nitrogen per 100 g dry matter was set for wheat starch, on theassumption that wheat protein would be the only source of nitrogen in starch(Codex Standard 118-1981).  The Codex Committee on Nutrition and Foods forSpecial Dietary Uses is developing a revised standard.  The current draft proposalwould define three categories of gluten-free foods: naturally "gluten free" (≤20 ppm of gluten), products that had been rendered gluten-free by processing (≤200 ppm), and any mixture of the two (≤ 200 ppm).  The Australia NewZealand Food Agency (ANZFA) defines gluten to mean "the main protein in wheat,rye, oats, barley, triticale and spelt relevant to the medical conditions,Coeliac disease and dermatitis hepetiformis."  ANZFA recognizes two classes offoods, gluten-free foods ("...no detectable gluten" and low-gluten foods ("...nomore than 20 mg gluten per 100 gm of the food") (ANZFA Food Code Standard1.2.8).  The Canadian standard for gluten-free is more general, simply statingthat "No person shall label, package, sell or advertise a food in a mannerlikely to create an impression that it is a "gluten-free" food unlessthe food does not contain wheat, including spelt and kamut, or oats, barley,rye, triticale or any part thereof" (Canadian Food and Drugs Act RegulationB.24.018).
IV. Discussion and RecommendationsA. General Approaches
Four general approaches were identified that could be usedto establish thresholds for allergens and glutens: analytical methods-based, safetyassessment-based, risk assessment-based, and statutorily-derived.  With any ofthese approaches, planned iterative re-evaluation of threshold values should becarried out as new knowledge becomes available.  These approaches aresummarized in Table IV-1 and described in detail below.
Table IV-1.  Approaches to EstablishingThresholds
**Type of Approach**

**Examples**

Analytical methods-based

Labeling of sulfiting agents

"Zero" tolerance policy for *Listeria monocytogenes*  in ready-to-eat foods

Safety assessment-based

Evaluation of food additive petitions

Risk assessment-based

Guidance levels for *Vibrio parahaemolyticus* in raw  oysters

Statutorily-derived

Labeling exemption for highly refined oil in the FALCPA

**1. Analytical Methods-Based Approach.**  In ananalytical methods-based approach, thresholds are determined by the sensitivityof the analytical method(s) that can be used to verify compliance.  Thiseffectively establishes a "regulatory threshold," although this threshold isnot necessarily correlated to biological effects.  This approach has been usedin food labeling.  For example, the requirement to declare sulfiting agents onproduct labels when foods contain 10 ppm or greater is based on the limit ofsensitivity of the analytical method used to measure these agents.

The issues that need to be considered when using ananalytical methods-based approach to establish a threshold include:
- What are the sensitivity and specificity of the method?
- Has the method been adequately validated?
- How will the method be used?
- How will the threshold be modified when improved methods are developed?

The strength of this approach is that it is relativelysimple, straightforward, and easy to implement.  However, it is appropriate touse an analytical methods-based approach to establish thresholds for allergensor gluten only if analytical techniques are available for all major foodallergens and celiac-associated glutens.

**2. Safety Assessment-Based Approach.** Safetyassessments are routinely applied to public health issues related to substancesin foods, such as chemical contaminants or food additives, particularly when abiological threshold can be justified scientifically.  The definition of"safe" varies according to the applicable legal provision.  For example, forcontaminants, the statutory definitions of safety are proscribed in section402(a)(1).  Food is considered adulterated if an added contaminant isin the food in a quantity "...which may render it [the food] injurious tohealth", or, if the substance in an inherent natural constituent of the food (i.e."not an added substance"), is in the food in a quantity that would "ordinarilyrender it [the food] injurious to health".   As another example, thephrase "reasonable certainty that no harm will result" is used in section408 (a)(4) regarding the safety of tolerances for a pesticide  chemical residue in or on a food.  

For a safety assessment, the term "safety" has connotationsinvolving both the degree of certainty and an assumption of "negligiblerisk."  The prototype chemical safety assessment is the Acceptable DailyIntake (ADI) method which was first articulated by Fitzhugh and Lehman (1954)for use in considering the significance of available animal data. This approachor variations of it are used throughout the world (WHO, 1987).  The ADIfor a chemical is calculated from the No Observed Adverse Effect Level (NOAEL)and Uncertainty Factor (UF) using the following equation:

ADI = NOAEL / UF.

The same basic methodology can be used to derive otherregulatory standards such as Tolerable Daily Intake (TDI), Reference Dose(RfD), and Minimal Risk Level (MRL).  These values are derived from controlledanimal studies, human clinical studies, or epidemiological studies that providethe exposure level for which there is not apparent or the lowest observableadverse effect (i.e., NOAEL, LOAEL). These adverse effect levels are alsoconsidered in conjunction with one or more uncertainty factor(s). Uncertaintyfactors are applied to account for inter-species and inter-individualdifferences and other uncertainties in the data (WHO, 2004).

There have been consistent efforts to improve this processto make better use of scientific knowledge.  These efforts have focused onboth replacing the NOAEL approach and refining the development ofuncertainty factors.  One example is the development of the benchmark dose(BMD) concept (Crump, 1984; Kimmel and Gaylor, 1988).  The BMD conceptinvolves fitting a dose-response model to all the available data and todetermine the statistical lower bound of the BMD (i.e., the BMDL). The major advange of the approach is that the BMDL is not constrainedto one of the experimental doses from a controlled study, as is the case withthe NOAEL (Crump, 1994).  EPA uses the BMD method in health riskassessments (Filipsson *et al*., 2003).

**3. Risk Assessment-Based Approach.**  A riskassessment is a systematic, scientific examination of known or potentialadverse heath effects resulting from human exposure to a hazard.  The generallyaccepted paradigm separates risk assessment into four components: hazardidentification, exposure assessment, hazard characterization (dose-response),and risk characterization.  This framework allows for organization of information,definition of uncertainties, and identification of data gaps.  Risk assessmentscan describe the likelihood of adverse health effects either quantitatively orqualitatively depending on the extent of the knowledge available, thecomplexity of the problem, and the time available to conduct the assessment.In quantitative risk assessments, risk is expressed as a numerical estimate ofthe chance of illness or death after exposure to a specific hazard.  Thisestimate represents the cumulative probabilities of certain events happeningand the uncertainty associated with those events.  A qualitative riskassessment, on the other hand, uses verbal descriptors of the risk anduncertainties, and often involves the aggregation of expert opinions.

Of the four approaches, the quantitative riskassessment-based approach is the most scientifically rigorous and providesinsight into the level of risk associated with specific exposures and thedegree of uncertainty inherent in the risk estimate.  An example of the use ofa risk estimate and associated uncertainty is the current standard forhypoallergenic infant formulas, where there is 95% certainty that 90% of thesensitive population will not react (Amer. Acad. Pediatrics, 2000).  The riskassessment-based approach is preferred when a biological threshold cannot bejustified scientifically.  Several recent papers have discussed the applicationof the risk assessment-based approach to food allergens (Bindslev-Jensen *etal*., 2002; Moneret-Vautrin and Kanny, 2004; Cordle, 2004; Wensing *et al*.,2002a).

The issues that need to be considered when using a risk assessment-based approach include:
- What is the biological endpoint or biomarker of concern?
- Is the response measurable?
- What is the population (or sub-population) of interest?
- What are the exposure levels?
- What data and assumptions are needed for the assessment,     and how do gaps in the existing data affect the level of uncertainty?

Other issues that should be considered in regard tounderstanding the relationship between the exposure level and nature of theresponse include:
- How sensitive and accurate are the available analytical     methods?
- How do changes in individual sensitivities over time and     within populations contribute to the overall uncertainty?
- What are the limitations of the clinical studies (e.g.,     small number of volunteers, not testing the most sensitive subpopulation)     that are used to determine the dose-response relationship and how do these     limitations contribute to the overall uncertainty?
- Which dose-response models (e.g., threshold,     non-threshold) are appropriate?

It is not clear whether the data and modeling techniquesavailable at the present time are sufficient to allow use of the riskassessment-based approach to establish thresholds for food allergens and for gluten.As an example of the complexity of this approach, the following describes theprocess of developing a dose-response model that can be used in a quantitativerisk assessment:

Steps in Developing a Dose-Response Model
- Determine the population of concern (e.g., infants,     children, pregnant women).
- Determine the endpoint or biomarker of concern (e.g.,     death, severe illness requiring hospitalization, subjective reactions such     as tingling of lip).
- Identify available relevant data including animal studies,     human clinical studies, and epidemiological data that relate dose to     frequency or severity of response.
- Select the appropriate dose-response model(s) that     characterize the shape of the dose-response curve.
- Fit the selected model(s) to the data.
- Characterize the uncertainty (i.e., curve weighting and/or     use of alternative plausible models).

**4. Statutorily-Derived Approach**.  Thestatutorily-derived approach establishes a threshold by extrapolating from anexemption established by Congress for another purpose.  For example, the FALCPAdefines "major food allergen" to include a food ingredient "thatcontains protein derived" from one of eight foods or food groups,"except... any highly refined oil" derived from one of those foods.  Ifconsumption of highly refined oils is not associated with allergic reactions,and if there is nothing unique about the proteins in highly refined oils, then consumptionof another food containing levels of protein that results in an exposure thatis equal to less than the level in a typical serving of highly refined oilsshould not be associated with allergic reactions. Thus, a threshold could beestablished for all food allergen proteins based on the level of protein inhighly refined oils.  There is no comparable statutory standard for gluten.
B. General Criteria for Evaluating and Selecting Approaches to Establish Thresholds
The general criteria used to evaluate the four approaches toestablish thresholds for allergens and gluten are shown in Table IV-2.Specific criteria related to food allergens are given in Section IV-C andgluten in section IV-D.  The specific criteria should be weighted appropriatelywhen implementing a particular approach.  The general criteria focus on dataavailability and data quality.  The Threshold Working Group recognizes thatscientific knowledge is the product of a process which is inherently imperfectand often incomplete.  As such, the degree of uncertainty in the data is a keyconsideration.  It is expected that any decisions on approaches forestablishing thresholds for food allergens or for gluten would requireconsideration of additional factors not covered in the current report.  Forexample, ease of compliance and enforcement, stakeholder concerns (i.e.,industry, consumers, and other interested parties), economics (e.g.,cost/benefit analysis), trade issues, and legal authorities are all significantfactors that are likely to influence the practicality of implementing anyapproach.  One option that is implicit in the following discussion of potentialapproaches is a decision not to establish thresholds at this time, at least forfood allergens.
Table IV-2.  General Criteria forEvaluating and Selecting Recommended Approaches to Establish Thresholds
**Criteria**

**Description**

Data Availability

Identification and review of currently available data that  can be used in any of the four approaches to establish a specific threshold.

Data Quality

Evaluation of the available data for utility,  completeness, and scientific soundness.  Evaluation of the degree of  uncertainty associated with the data.

**1. Feasibility**.  The published and unpublishedliterature summarized in Sections II and III of this report were reviewed todetermine the availability of the specific types of data needed for each of theapproaches to establish thresholds.  When necessary information was notavailable, the following questions were used to evaluate the existinginformation:  
- Is there surrogate or alternate information available that     could be used?  
- Is the existing knowledge sufficient to support reasonable     assumptions when specific data are not available?  
- What is the level of uncertainty associated with these     data and assumptions?

**2. Uncertainty**.  Uncertainty is typicallythought to arise from the lack of data or information.  Other sources ofuncertainty are often considered to be relevant to scientific evaluations suchas subjective judgment, statistical variation, sampling errors, and inherentrandomness (Byrd and Cothern, 2000).  Techniques are available to account foror measure some of these uncertainties.  For example, the uncertainty in adose-response model can be characterized using advanced techniques, such asmodel weighting, that measure the degree of credibility associated with themodel results (Carrington, 1997).  State-of-the-art food safety risk assessmentmodels, such as the HHS/FSIS *Listeria monocytogenes* risk assessment forready-to-eat foods (HHS/FSIS, 2003) also used techniques that separateuncertainty from biological variability.  It is important to note thatuncertainty is different from variability.  Uncertainty reflects incompleteknowledge about a system or population which can be reduced with additionalstudy.  Variability reflects the fact that all systems or populations have inherent,biological heterogeneity that is not reducible through further measurement orstudy (Voysey *et al*., 2002).  Sufficient knowledge is needed to accountfor both variability and uncertainty in order to evaluate the four approachesfor establishing thresholds.

As described above, uncertainty factors are used in safetyassessment calculations.  Fitzhugh and Lehman (1954) originally proposed asingle safety factor of 100-fold applied to animal data.  The justification forthis factor included both scientific issues and social values.  The scientificissues included the possibility that humans may be more sensitive to chemicalsthan the rodents used in laboratory tests and that there may be substantialvariability among individuals in a population.  In general, as uncertaintyincreases, the uncertainty factor employed in a safety assessment shouldincrease proportionally.  As a matter of practice, uncertainty is notcharacterized in a safety assessment, either formally or subjectively, as isdone in a quantitative risk assessment.  A minimum uncertainty factor of 10 isgenerally used to account for variation within the population when relying onhuman data and additional uncertainty factors may be included as appropriate.For example, the Food Quality andProtection Act (FQPA) of 1996 requires, incertain cases, a 10-fold factor in addition to any other uncertainty factors toprotect infants and children from exposure to pesticides .The assignment of uncertainty factors should be based on science but typicallywill include the application of expert judgment.

**3. Data Quality**.  The FDA Information Quality Guidelineswere used in evaluating the scientific data contained in this report.  Theseguidelines describe policies and procedures for ensuring the quality of theinformation disseminated by FDA.  In these guidelines, data quality is definedin terms of utility, objectivity, and integrity.  Utility is defined as theusefulness of the information to its intended users; objectivity aspresentation of the data in an accurate, clear, complete, and unbiased manner;and integrity as protecting the information from unauthorized access orrevision.  In particular, the guidelines provide transparency standards andensure clarity.
C. Allergen Thresholds: Evaluation and Findings
This section provides an evaluation of the data needed toestablish thresholds for the major food allergens.  Based on the availabilityand quality of the data, the Threshold Working Group provides findings that canbe applied to establish such thresholds.

**1.  Evaluation of Data Availability and Data Quality**

**a.****Sensitive Populations**.  Most clinicalstudies exclude patients who have had previous anaphylactic reactions or whohave very high IgE titers.  This suggests that the most sensitive individuals withinthe allergic population may be systematically excluded from these studies.Therefore, it is possible that the distribution of doses reported to elicit the"initial objective symptoms" is higher than would be expected if the entire allergicpopulation was considered.  The observed dose distribution may also not berepresentative of the allergic population in studies that use patientpopulations that are not known to be allergic to the food being tested (e.g.,testing milk allergic patients for sensitivity to soy).  In addition,individual sensitivity varies over time.  This means that "high sensitivity"may be a transient condition for an individual.

There are a number of reports in the scientific literaturedocumenting unusual allergic reactions in individuals.  These case studiesinclude reactions to incidental exposures to allergens.  These reports aredifficult to interpret because the level of exposure and potential influence ofother factors (e.g., medications, exercise) are not known.  Nevertheless, ifthese reports document true allergic reactions, this suggests that theseindividuals could be considered to be highly sensitive when compared to thegeneral population of food allergic individuals.

Based on currently available data, the Threshold WorkingGroup was unable to identify any scientifically-based studies that indicatethat the standard 10-fold uncertainty factor used in safety assessments forinter-individual variability is not adequate to account for variation withinthe sensitive population.  However, because of the limitations in the clinicalstudies and the case reports discussed above, this assumption should bereexamined as more data on the distribution of sensitivities within thepopulation become available.

**b.****Biomarkers**.  Because a number of differentsymptoms are associated with allergic reactions, it is important to identifythe most appropriate biological markers for establishing thresholds.  Thesymptoms of an allergic reaction can be either subjective (reported by thepatient but not overtly measurable) or objective (overt reactions that areobserved or measured by another person).  Objective symptoms vary on acontinuum of severity from mild rashes to fatal anaphylaxis.  Although each ofthese symptoms is an "adverse effect," there is no consensus where on thiscontinuum the symptoms become "serious adverse effect."  This makes itdifficult to apply either risk assessment- or safety assessment-basedapproaches to establish thresholds for food allergens because both approaches requirethat the adverse end point be well defined.

Most clinical studies expose patients to increasing doses ofan allergen until the first objective symptom is observed.  This is often, butnot always, a relatively mild symptom.  For ethical and technical reasons, fewstudies measure dose-response relationships for individual patients beyond theinitial objective symptom.  Therefore, the currently available literatureprovides data based on the "initial objective symptom."  Although the "initialobjective symptom" is the biomarker measured in most available allergen clinicalstudies, it is unclear whether these symptoms are consistently consideredacross these studies.  It is also not clear whether and when subjective reactionsshould be considered  "adverse effects," or should influence the selection of aNOAEL or LOAEL for safety assessments.

Normally, the use of the "initial objective symptom" wouldlead to threshold values that are "protective" in relation to the overall riskto food allergic consumers.  However, it should be noted that severe reactionshave been reported as the initial objective symptom in some cases.  Forexample, Perry *et al*. (2004) reported that "reaction severity did notincrease as the amount of challenge food ingested increased."  Likewise, theonly severe reaction observed by Hourihane *et al*. (1997) in a populationof 100 patients occurred at the lowest dose tested.  However, considering thatthe use of the "initial objective symptom" does appear to be generallyprotective, and that such data would be used in conjunction with appropriateuncertainty factors, it may not be necessary to differentiate among "mild," "serious,"or "life-threatening" symptoms when establishing a safety assessment-basedthreshold from existing clinical data.

**c. Analytical Methods for Food Allergens.**  Thecriteria used to evaluate the available analytical methods for the major foodallergens are shown in Table IV-3 and are applied in Appendix 1.
Table IV-3.  Specific Criteria for EvaluatingAnalytical Methods for Food Allergens  Criteria  Comments  1. Has the method been validated?     Methods that have been validated (such as by AOAC) are  preferred.  Alternatively, the sensitivity, precision, and reproducibility of  the method has been demonstrated in a peer reviewed publication.   2. Is the method sufficiently sensitive?   The limit of detection and the limit of quantitation  should be below the levels that appear to cause biological reactions.   3. Does the method detect both raw and processed food  allergens?   The relevant processing methods (e.g., boiling, roasting,  retorting) will depend on the food.   4. Has the species specificity of the method been  determined?   This is most relevant to methods for allergens such as  fish and tree nuts.   5. Has the protein target (or targets) for the method been  determined?   This is relevant to determining whether the assay detects  specific allergenic proteins or general biomarkers.   6. Is the method practical?   The method should use common laboratory equipment and  supplies. 
The response of sensitive consumers to exposure to anallergen is dependent on the levels of the allergen in the food and the amountof food consumed, two factors for which there is both variability anduncertainty.  The levels of allergen in foods may not be known for a number ofreasons, particularly when the presence of the allergen is the result ofcross-contact.  Even in highly controlled clinical studies, questions regardingthe level of allergen arise due to differences in the methods used to processand prepare the test material, incomplete characterization of this material,variability in allergen levels among different sources of the food, lack ofstandardized reference materials, and differences in the analytical methods usedto quantify the levels of the allergen.

The methods used to quantify and express the doses receivedduring clinical studies and adverse event investigations are not consistent,and this increases the uncertainty associated with the available data.  Theamount of an allergen consumed has been described in terms of total weight of afood consumed, total protein from an allergenic ingredient, or amount ofspecific allergenic proteins.  Although the last description is scientificallythe most accurate, it is also the most difficult to use because not allindividuals are allergic to the same proteins in a food allergen and all theallergenic proteins may not have been identified for a particular food.Measurements based on the whole foods are simple, but increase the level ofuncertainty because the composition of the food may vary.  For example, changesin water content of a food would change the relative amount of allergenicprotein present in serving sizes of a specified mass.  Further, the amount of proteinpresent as a percent of the total weight of the food may vary due toenvironmental factors, seasonal factors, production variability, or betweendifferent cultivars or strains.  The Threshold Working Group recognized that thescientifically most accurate means of assessing exposure would be to quantifyindividual allergenic proteins, but concluded that the most practical approachfor evaluating the currently available data is to measure exposure in terms ofthe total protein from a food allergen.  This is also consistent with currenttechnology for detecting food allergens.

It should also be noted that, while clinical exposures areexpressed in terms of doses (i.e., g, mg, or μg), allergen levels in foodsare actually measured as concentrations (i.e., ppm, percent, or mg/kg).  Thesevalues can be related by defining a standard serving size, usually 100 g.However, it is well documented that the actual serving eaten by consumersshould be treated as a variable and a source of uncertainty when assessing foodrelated exposures.

**d. Challenge Studies.**  Clinical food challengestudies are recognized to be the most accurate way to diagnose allergies and tomeasure sensitivity to an allergen (Sampson, 2005).  Unfortunately, the designof these food challenge studies varies widely.  The lack of standard protocols,variations in the dosing regimes (including number of doses, the intervalbetween doses, and the relative size of the doses), and differences in the foodsources (including differences in preparation and presentation) result inuncertainties when comparing the results of different studies.  Double blindplacebo controlled food challenges (DBPCFC) are considered the most robustclinical studies and data from these studies should be given preferencewhenever they are available.  Food challenge studies are generally not designedto determine a lack of reaction (i.e., NOAEL).  Instead, the doses that producepositive allergic reactions are generally reported, providing an estimate ofthe LOAEL for the population being studied.  Despite the uncertaintiesassociated with food challenge data from the literature, LOAELs from humanclinical trials currently provide the best data for estimating population-basedreactions to food allergens.  In a safety assessment-based approach, the use ofLOAELs instead of NOAELs would introduce additional uncertainty.  A standardDBPCFC protocol has been proposed to identify NOAELs for various foodallergens, but few publicly available, peer-reviewed data of this nature areavailable at this time.  The specific criteria used to evaluate food challengestudies are shown in Table IV-4, and applied in Appendix 2.
Table IV-4.  Specific Criteria for EvaluatingAllergen Oral Challenge Studies  Criteria     Comments     1. Has the study been published in a peer-reviewed journal?      Published, peer-reviewed studies are preferred although  unpublished studies may be considered.    2. Were the criteria for selecting the test population  clearly and completely described, and are they appropriate?      This information is needed to evaluate how the study  results apply to at-risk populations (i.e., was the tested population  allergic to the tested food?).    3. Was the test material clearly and completely described?    This information is needed to determine the amount of  allergenic protein in the test material.    4. Was the lowest tested dose of allergen described, or  can it be calculated?      This information is needed to determine a NOAEL or LOAEL.     5. Were the total number and progression of dose levels described,  or can they be calculated?  (i.e., can the entire dose series be explicitly  determined?)    This information is not needed for a safety assessment,  but is needed for a risk assessment.    6.  Did some of the test population respond to the lowest  dose?      NOAELs and LOAELs cannot be determined in studies in which  reactions occurred at the lowest dose tested.    7. Were the allergic reactions observed clearly described?    Objective reactions are preferred for both safety and risk  assessments     8.  Were the data sufficient to describe the dose-response  pattern for the population tested (e.g. for determining a cumulative dose-response  curve)?    This information is needed for a risk assessment.  
**e.  Differences Among Food Allergens.**  Allergensdiffer widely both in their potential to elicit allergic reactions and in theseverity of these reactions.  The simplest approach to dealing with thesedifferences would be to establish a single threshold based on sensitivities tothe most potent allergens.  This threshold is likely to be unduly restrictivefor many allergic consumers.  Alternatively, separate thresholds could beestablished for each food allergen.  However, the data needed for the separatethreshold approach are not available for many allergens.  The Threshold WorkingGroup concluded that, to the extent possible, each food allergen should betreated independently but that a single threshold should be established if independenttreatment is not possible. If a single threshold is established, it could bebased on the allergenic food that elicits an allergenic reaction at the lowesttotal protein level.

Some of the major allergens identified in the FALCPA consistof multiple species (i.e., tree nuts, fish, crustacean shell fish).  Becauseconsumers who are sensitive to one species in a group are also likely to besensitive to other members of the group, the Threshold Working Group concludedthat any thresholds established for these allergens should be based on thecombined amount of these species present.

**f.  Processing Effects.**  Most of the food allergens identifiedin the FALCPA are eaten in a processed form.  The existing data show thatprocessing can increase, decrease, modify, or have no affect on allergenicitydepending on the allergen, the process, and the matrix involved.  A processthat modifies the structure of an allergenic protein could reduce allergenicityfor one population of susceptible individuals while simultaneously increasingallergenicity for a separate susceptible population.

Most clinical studies are conducted using test materialsthat have been processed, such as peanut butter prepared from roasted peanuts.Therefore, these studies are likely to mimic actual consumer exposure to theallergen.  However, some uncertainty remains because consumers are exposed tofood allergens processed in many different ways.  It would not be practical toconduct the large number of clinical studies that would be necessary to reducethis uncertainty.  Fish appears to be an important exception because raw fishis often used as a test material.  Most people eat cooked fish and this shouldbe taken into account when evaluating the results of these studies.

**2.  Options and Findings**

There are four general approaches that could be used toestablish thresholds for food allergens -analytical methods-based, safety assessment-based, risk assessment-based, and statutorily-derived.Each approach has strengths and weaknesses, and the application of each islimited by the availability of appropriate data.  It is likely that that therewill be significant scientific advances in the near future that will address anumber of the limitations identified in this report.  The Threshold WorkingGroup was aware of several potentially important studies that are currently inprogress, but was unable to evaluate them because the data or analyses wereincomplete.

**Finding 1**.  The initial approach selected to establish thresholds for major food allergens, thethreshold values, and any uncertainty factors used in establishing thethreshold values should be reviewed and reconsidered periodically in light ofnew scientific knowledge and clinical findings.

**a.  Analytical Methods-Based Approach.**  The analyticalmethods-based approach could be used to establish thresholds if the availabledata are insufficient to establish thresholds using one of the otherapproaches.  This approach requires that analytical methods be available todetect all allergens. Thresholds would be defined by the limits of detection ofthe available analytical methods, but there would be no relationship betweenthese thresholds and the biological response thresholds.  Currently, the lowerdetection limits for commercially available allergen ELISA or immunoassay testkits are in the range of 0.1 to 1.0 �g protein/g of food, but such kits are notavailable for all food allergens. Establishing thresholds at levelshigher than the lower detection limits of the analytical methods would requirethe use of assumptions about the biological response thresholds.  In that case,the thresholds are actually based on using one of the other three approachesand should not be considered an analytical methods-based threshold.

*Advantages*.  When accurate, validated methods areavailable to measure food allergens, determining a threshold based on thesemethods can be a straightforward way to establish that products are incompliance with this defined level.

*Limitations*. There are several disadvantages tousing this approach in determining thresholds for food allergens:    
- The approach is not risk-based and it is likely that the     appropriateness of any thresholds established using this approach will be     questioned as existing methods are improved or new methods are developed.     Further, in the absence of information on biological response thresholds,     it is difficult to assess how well thresholds established using this     approach protect public health.
- Validated analytical methods are currently not available for     all the major food allergens.  However, this is likely to change rapidly     if there is a need for such analytical capability.  
- There is uncertainty as to the performance of the     available analytical methods in the wide variety of food matrices that are     likely to be encountered.  Theoretically, the test methods should be     validated for all foods and food matrices, but this is not practical.  
- Current methods, which are based on a food's total protein     content, will not be sufficient in the future if techniques and     technologies for reducing the levels of specific allergenic proteins are developed.

Presumably, the analytical methods used to establishthresholds in this approach could also be used to evaluate compliance with anyapplicable legal requirements.  However, the ability to use these methods to helpprevent the introduction of unlawful product into the market place would requirethat the methods be applied in a scientifically supportable manner.  This wouldrequire the establishment of a statistically supportable sampling plan.  Thecost of the sampling to a degree sufficient to provide reasonable statisticalconfidence is potentially an issue.

**Finding 2.** Theanalytical methods-based approach could be used to establish thresholds forthose food allergens for which validated analytical methods are available.However, if this approach is used, the thresholds should be replaced bythresholds established using one of the other approaches as quickly aspossible.

**b.  Safety Assessment-Based Approach.**  The safety assessment-based approach could be used to establish thresholdsbased on NOAELs or LOAELs reported in the literature in combination withappropriate uncertainty factors.  Because very few publications report NOAELsor present results in a form that allows NOAELs to be calculated, this type ofanalysis would, for most food allergens, be based on LOAELs.  NOAELs should beused when they are available or can be calculated (see Appendix 2). 

As discussed previously, there are substantial differencesin the relative potency of different food allergens (e.g., peanut vs. soy).  Asnoted in Appendix 2 and summarized in Table IV-5, the reported LOAELs forpeanuts are considerably lower (maximum of 10 mg protein) compared to soy(maximum 522 mg protein).  A single threshold for food allergens, based on themost potent food allergens, could be employed if, as a matter of riskmanagement policy, a single threshold is considered desirable.  However, thiscould be considered overly protective, particularly in the case of soy.
Table IV-5.  Summary of PublishedLOAELs for Food Allergens  Food    Range of LOAEL (mg protein)    Egg    0.13 to 1.0    Peanut    0.25 to 10    Milk    0.36-3.6    Tree Nuts    0.02-7.5    Soy    88-522    Fish    1-100  
*Advantages*. Calculation of threshold levelsbased on NOAELs or LOAELs and the application of appropriate uncertaintyfactors to estimate exposure is relatively straightforward.  When there arelimited data in the literature, the application of appropriate uncertaintyfactors provides confidence that the majority of the sensitive populations willbe protected.  For a number of the major food allergens, there is reasonablygood agreement among the reported LOAEL values.  Establishing thresholds usingthe safety assessment-based approach and currently available clinical data hasthe advantage of being directly linked to biological effects.

*Limitations*. There are limited clinical trialdata for most allergens and most available clinical food challenge studies havenot been designed to identify a NOAEL.  Furthermore, an inherent, butunexamined, assumption in all clinical studies is that the reactions seen in aclinical setting are representative of the reactions to food allergen exposurethat occur in the real world.  Most available clinical data are primarilylimited to identifying LOAELs, and there is no way to know whether doses belowthe observed LOAEL would still elicit a reaction.  Thus, the selection ofappropriate factors to account for uncertainty and inherent variability iscritical in using the safety assessment-based approach.  Until there is a consensusas to whether subjective symptoms are acceptable biomarkers or which objectivesymptoms are considered harmful, it appears prudent to consider as adverse anyobjective reaction observed in a clinical trial.

We have identified several data gaps for allergens that addto the uncertainty associated with setting thresholds.  Critical areas ofuncertainty and variability include:
- Intraspecies differences.  Safety assessments typically     apply a 10-fold uncertainty factor to account for the variability both     between individuals and variability in responses for a particular     individual.
- Sensitive population of interest.  The existence and size     of highly sensitive sub-populations of allergenic individuals and their     lack of participation in reported clinical trials is a potential data gap     and should be included in the uncertainty factors.  It is unclear whether     the standard 10-fold uncertainty factor for variability within a species     is sufficient to account for potential highly sensitive subpopulations.     Because of the potential severity of reaction for this subpopulation     (i.e., lethality) it seems prudent to include an additional margin of     safety (e.g., a 10-fold uncertainty factor) for this uncertainty.  It is     not unusual for safety assessments to provide additional protection for     susecptable populations.  For example, EPA uses an additional safety     factor in re-evaluating pesticides as per the Food Qualtiy Protection Act     (FQPA, 1996) to account for the greater susceptibility of children to     certain pesticides.
- Adequacy of clinical trial data.  Most of the available     data from clinical trials report LOAELs.  There is uncertainty associated     with using LOAELs rather than NOAELs to establish a threshold.  For peanuts,     one of the few food allergens for which NOAEL values are available, the     observed LOAELs for objective symptom are approximately 2 to 3 fold greater than the observed NOAELs.  
- Other.  Additional data gaps have been identified by the     Threshold Working Group; however, we concluded that uncertainties     associated with these factors were not sufficient to warrant additional     uncertainty factors.  These data gaps include the following: (1) the use     of total protein from a food as a surrogate for measuring the level of     specific allergenic proteins in clinical trials; (2) variability in     serving sizes and related exposure factors, and (3) the effects of food     processing on the levels and reactivity of allergenic proteins is     incompletely defined.  

The Threshold Working Group acknowledges that it isdifficult to estimate uncertainty factors that apply in all situations for allallergen threshold determinations when using a safety assessment-basedapproach.  We can, however, assume that a standard uncertainty factor of 10-foldshould be applied for intraspecies differences in humans.  Additionaluncertainty factors could be added if justified from data gaps.  In Table IV-6we use peanuts, widely considered to be among the most potent food allergens,to illustrate how specific uncertainty factors may be developed for use in asafety assessment-based approach to set a threshold if that approach isadopted.
Table IV-6.  Example of UncertaintyFactors for the Safety-Assessment-Based Approach Using Peanuts.
**Description**

**Uncertainty  Factor**

**Justification**

Intraspecies difference1

10

Standard factor for intraspecies variability

Estimation of NOAEL2

Not applicable

Two studies were identified that report NOAELs

Sensitive population3

10

Used to account for additional margin of protection for  more susceptible populations not included in clinical trials

**Overall Uncertainty Factor for Peanuts = 100**

1 This includes both between- and within-individualvariability.

2 This includes both a factor for converting the LOAELto a NOAEL and an additional factor for the uncertainty associated with thatconversion.  In this example for peanuts, there are data on both subjective andobjective NOAELs and LOAELs.  If the NOAEL values are used, the uncertaintyfactor is 1-fold (i.e., not applicable).  If the LOAELs had been used, thisvalue would have been higher. If subjective symptoms observed at lower levelsare used, a different uncertainty factor may be considered.

3 This includes uncertainty associated with anadditional margin of protection to account for the potential severity of reaction(e.g., lethality) for the highly sensitive subpopulation.

**Finding 3**.  Thesafety assessment-based approach, based on currently available clinical data,is a viable way to establish thresholds for food allergens.  If this approachis employed, the LOAEL or NOAEL determinations used should be based on evidenceof the "initial objective symptom."  Individual thresholds should beestablished for each of the major food allergens. If it is not feasible toestablish individual thresholds, a single threshold based on the most potentfood allergens should be established.  In those instances where a LOAEL is usedrather than a NOAEL to establish a threshold, an appropriate uncertainty factorshould be used.

**c.  Risk Assessment-Based Approach.** The useof the risk-assessment-based approach requires analysis of the populationdistributions of allergic sensitivities for each of the major food allergens.These distributions would then be used in conjunction with data on exposures toassess the probability of an adverse effect.  These distributions could also beused to evaluate the likely efficacy of different risk reduction strategies.

*Advantages*. The quantitative riskassessment-based approach is the most scientifically rigorous approach andprovides the most insight into both the level of protection and the degree ofuncertainty associated with an exposure level.  Several recent publicationsthat present preliminary quantitative risk assessments based on data fromclinical trials suggest that this approach shows promise (Bindslev-Jensen *etal*., 2002; Moneret-Vautrin and Kanny, 2004; Cordle, 2004; Wensing *et al.,*2002a).

*Limitations*. Quantitative risk assessmentsrequire the most data of any approach to establish thresholds for foodallergens, because they are based on determining the entire dose-responsecurve, not simply a NOAEL or LOAEL.  The data currently available in theliterature for food allergens are generally not detailed enough to be usefulfor quantitative risk assessment.  Further, the underlying mathematicalprocedures and assumptions have not been fully described for the models thathave been published.  No consensus has been reached regarding the mostappropriate mathematical model to use for analyzing allergen reaction data.

**Finding 4**. Of the four approaches described, the quantitative risk assessment-based approachprovides the strongest, most transparent scientific analyses to establishthresholds for the major food allergens.  However, this approach has onlyrecently been applied to food allergens, and the currently available data arenot sufficient to meet the requirements of this approach.  A research programshould be initiated to develop applicable risk assessment tools and to acquireand evaluate the clinical and epidemiological data needed to support thequantitative risk assessment-based approach.  Thresholds established using thisapproach should be reevaluated periodically as new data and tools becomeavailable.

**d.  Statutorily-Derived Approach.**  As discussedabove, an allergen threshold could be extrapolated from a statutory exemptionestablished by Congress for another purpose, such as the FALCPA exemption for"highly refined oils."  Thus, a threshold could be established for all foodallergen proteins based on the level of protein in highly refined oils.

There are surprisingly few data available in the publishedscientific literature reporting on the levels of proteins in highly refinedoils.  The criteria used to evaluate studies measuring protein levels in foodoils are shown in Table IV-7 and applied in Appendix 3.
Table IV-7.  Specific Criteria forEvaluating Protein in Oil Studies
**Criteria**

**Comments**

1.  Has the study been published in a peer-reviewed journal?

Published, peer-reviewed studies are preferred, although  unpublished studies can be considered.

2.  Was the oil completely described, including all  refining and treatment steps?

The level of processing must be known both to compare  values among studies and because each processing step may change the level of  protein in oil.

3.  Was the method used to extract the protein completely  described?  

Extraction procedures should be described in sufficient  detail to allow the extraction to be reproduced and, ideally, extraction  efficiencies should be measured and reported.

4.  Was the method used to quantify protein levels  completely described? 

The lack of these data increases the level of uncertainty

5.  Were replicate samples or batches tested, and was  there a statistical analysis of these data?

The lack of these data and statistical analysis increase  the level of uncertainty.

Based on the data presented in those studies that reportedlevels other than "not detected", the overall range of protein concentrationsfor highly refined oils was 0.014 to 16.7 �g protein/ml oil, with a mean of2.35 �g/ml.  The combined mean protein concentration for the two most widelyused oils derived from food allergens, soy and peanut, is 0.74 �g/ml with astandard deviation (std) of 1.3 �g/ml.  A threshold could be based on the meanprotein concentrations or on the mean plus some multiple of the standarddeviation.  For example, using the mean protein concentrations for peanut andsoy oils, protein levels for the mean, mean + 1 std, mean + 2 std, or mean + 3std would be the 0.74, 2.05, 3.36, and 4.67 ug/ml, respectively.

*Advantages*.  The primary advantage to the statutorily-derivedapproach is that it is derived from FALCPA's exemption for highly refined oilsfrom labeling provisions in the FALCPA.

*Limitations*.  The primary limitation of this approachis that it is based on an extrapolation of a level derived from a statutoryexemption rather than a rigorous, systematic evaluation of all the available scientificdata.  Because not all the eight major food allergens are used to producehighly refined oil, the use of a statutorily-derived threshold for all food allergenswould be based primarily on the protein levels in highly refined soy or peanutoil.  Another current significant limitation is the lack of data on the levelsof protein in highly refined oils.  Based on the data that are currentlyavailable and estimates of the amount of oil consumed as a food or foodingredient, it is likely that a threshold based on this approach would be unnecessarilyprotective of public health.

**Finding 5.** The statutorily-derived approach provides a mechanism for establishingthresholds for allergenic proteins in foods based on a statutory exemption.Potentially, this approach could be used to set a single threshold level forproteins derived from any of the major food allergens.  This approach mightyield thresholds that are unnecessarily protective of public health compared tothresholds established using the safety assessment-based approach.  However,confirming this would require additional data.  If this approach is employed toestablish thresholds, it should be used only on an interim basis and should bereevaluated as new knowledge, data, and risk assessment tools become available.
D. Gluten Threshold: Evaluation and Findings
Section 206 of the FALCPA requires that the term"gluten-free" be defined for use on food labels.  The law neitherdescribes how gluten-free should be defined nor states whether there is a safelevel of gluten.

This section provides an evaluation of the available data tosupport various approaches for establishing a threshold for gluten.  Athreshold, if established, could be the basis for decisions on whether to usethe term "gluten-free" on product labels.

**1.  Evaluation of Data Availability and Data Quality**

**a.  Sensitive Populations**.  Like food allergies,celiac disease affects only a small part of the U.S. population (estimated at1%).  Susceptibility to celiac disease is genetically determined and is linkedto the presence of the DQ2 or DQ8 HLA alleles.  However, carrying these allelesdoes not necessarily lead to celiac disease.  Both acute and chronic morbidityhave been well documented for individuals with symptomatic celiac disease.  Agluten-free diet has been shown to greatly reduce the risk for cancer andoverall mortality for these individuals.  The potential benefit of agluten-free diet has not been established for individuals with silent or latentceliac disease.

**b.  Biomarkers**.  Biomarkers of genetic susceptibilityand gluten exposure which allow for non-invasive diagnosis of individuals withceliac disease have been defined.  Examples of these biomarkers includecirculating antibodies for gliadin (AGA), endomysial (EMA), and tissue transglutaminase(tTG), each of which can be monitored in individuals carrying the DQ2 and/orDQ8 HLA alleles.  In asymptomatic individuals with these genetic markers, thepresence of anti-EMA or anti-tTG antibodies indicates the presence of latentceliac disease.  However, intestinal mucosal inflammation may occur long beforethe development of clinical symptoms or a rise in antibody titers following agluten challenge.  Further, antibody titers have not been shown to correlatewith disease severity.  Therefore, for asymptomatic individuals, intestinalbiopsies are required to evaluate disease activity or severity.  Biopsies areinvasive, associated with false negatives, and impractical for frequentmonitoring of disease activity or severity.

**c.  Foods of Concern**.  The foods of concern forindividuals with, or susceptible to, celiac disease are the cereal grains thatcontain the storage proteins prolamin, gliadin and glutelin (commonly referredto as glutens in wheat), including all varieties of wheat (e.g., durum, spelt,kamut), barley (where the storage proteins are called hordiens), rye (where thestorage proteins are called secalins), and their cross-bred hybrids (such astriticale).  A small percentage (5% to 10%) of individuals with celiac disease isalso sensitive to the storage proteins in oats (avenins).

**d.  Methods of Analysis**.  The criteria used toevaluate the available methods of analysis for gluten in food are shown inTable IV-8 and are applied in Appendix 4.  A number of commercial immunology-basedELISA test kits for the detection of gluten in foods are available, and one hasbeen validated by AOAC (the Tepnel kit, validated at 160 ppm).  One limitationof these kits is that they only detect prolamins. This is not likely to limitthe detection of gluten in foods because prolamins and glutelin occurtogether.  However, it may lead to an underestimate of the level of glutenpresent.  Also, none of the test kits cross-reacts with protein extracts fromoats, which limits their efficacy for the small portion of celiac patients whoare also sensitive to oats.  This would require the development of test kitssuitable for the detection of oats.
Table IV-8.  Specific Criteria forEvaluating Gluten Analytical Methods
**Criteria**

**Comments**

1. Has the method been  validated?  

Methods that have been  validated (such as by AOAC) are preferred.  Alternatively, the sensitivity,  precision, and reproducibility of the method should have been demonstrated in  a peer reviewed publication.

2. Is the method sufficiently  sensitive?

The limit of detection and the  limit of quantitation should be below the levels that appear to cause  biological responses in most patients with celiac disease.

3. Are extraction methods  available for both raw and baked foods?

Different methods may be  needed; each should be validated.

4. Does the method measure  proteins from all relevant foods?  

The cereal grains associated  with celiac disease include wheat, barley, rye, and their cross-bred  hybrids.  Oats may be of concern for some celiac patients.

5. Does the method measure  both gliadins and glutenins?  

The storage proteins in cereal  grains (generally referred to as gluten) include both prolamin proteins  (gliadins) and glutelin proteins (glutenins).  Ideally, both of these should  be measured.

6. Is the method practical?

The method should use common  laboratory equipment and be reasonably priced.

**e.  Oral Challenge Studies**.  The criteria used toevaluate the available gluten oral challenge studies are provided in Table IV-9and applied in Appendix 5.  Only a limited number of gluten or gliadinchallenge studies have been conducted.  Of these, most have monitored thesubjects' acute responses to a single high dose of gluten or gliadin.  Theseacute studies were not designed to establish a NOAEL or (in most cases) aLOAEL, and the results are not directly applicable to the chronic, low levelexposures that may lead to celiac disease.  Most clinical studies only test oneor two doses.  In addition, many studies monitor only biomarkers of glutenexposure (humoral and cell-mediated immune system activity) instead of usingbiopsies to measure intestinal damage in response to gluten in the diet.  Basedon the criteria in Table IV-9, two currently available studies are consideredto be of high utility.  The data in these studies can be used to calculateLOAELs for acute high dose exposures.  There are essentially no data availableon the impact of chronic consumption of lower gluten levels.
Table IV-9. Specific Criteria forEvaluating Gluten Oral Challenge Studies
**Criteria**

**Comments**

1. Has the study been  published in a peer-reviewed journal?

Published, peer-reviewed  studies are preferred although unpublished studies may be considered.

2. Were the criteria for  selecting the test population clearly described in the study?

This information is needed to  evaluate how the study results apply to the at-risk population.

3.  Was the tested food  material clearly and completely described?

It is important to know the  level of gluten in the test material.

4.  Was the dose regime  clearly and completely described?

A study designed to measure  chronic exposure (low doses over a long period of time) is preferable.  Extrapolation of long-term effects from short term studies increases the  level of uncertainty.

5. Were the criteria for  characterizing responses clearly described?  

This information is needed to  evaluate the relevance of the response measured.  A definitive diagnostic  assessment showing clinical symptoms or intestinal mucosal changes compared  to controls is preferred.

6. Are the raw data  (individual dose and responses) available for each individual tested?

These data are needed to  develop a risk assessment-based dose-response model.

**2. Options and Findings**

The feasibility of using each of the four methods toestablish a threshold for gluten was evaluated in light of the available data.As with food allergens, it is likely there will be significant scientificadvances in the near future that will address a number of the limitationsidentified in this report.  The Threshold Working Group is aware of severalpotentially important studies that are currently in progress, but we are unableto evaluate them because the data or analyses are incomplete.

In particular, the Threshold Working Group is aware ofunpublished data from an ongoing clinical trial of the subchronic effects ofgluten on celiac patients.  The "Italian Microchallenge Study" is utilizingintestinal biopsies to relate changes in the intestinal mucosa to antibodybiomarkers (Fasano, 2005 personal communication).  Preliminary results indicatethat daily consumption of both 10 mg and 50 mg of dietary gluten were welltolerated after three months of continuous consumption, but that minimalhistological changes were seen in patients consuming 50 mg of gluten daily.Because these data have not yet been published, these results were notconsidered further.

**Finding 6**.  Theinitial approach selected to establish a threshold for gluten, the thresholdvalue selected, and any uncertainty factors that were used to establish thethreshold should be reviewed and reconsidered periodically in light of newscientific knowledge and clinical findings.

**a.  Analytical Methods-Based Approach**.  As with foodallergens, an analytical methods-based approach could be used to establish athreshold for gluten if the available clinical and epidemiological data areinsufficient to use one of the other approaches.  This approach requires thatanalytical methods be available to detect all relevant glutens.  Thresholds aredefined by the limits of detection of the available analytical methods, butthere is no relationship between these thresholds and the biological responsethresholds.  Currently, the lower limit of detection for the commercially availablegluten test kits are in the range of 10 �g gluten/g of food.  Establishingthresholds at levels higher than the lower detection limits of the analyticalmethods requires the use of assumptions about the biological responsethresholds.  In that case, the thresholds are actually based on using one ofthe other three approaches and should not be considered an analyticalmethods-based threshold.

*Advantages*.  A threshold established using theanalytical methods-based approach can easily be incorporated into any applicableFDA compliance programs that combine a  specific standard method with astandardized sampling scheme.

*Limitations*.  Several factors limit the applicabilityof the analytical methods-based approach to establish a threshold for gluten.At this time, only one commercially available analytical method has beenvalidated, and that method was validated for detection at a relatively highconcentration of gluten.  In addition, there are limited data on theperformance of the available methods in the wide variety of food matrices thatcould potentially contain gluten.  Therefore, further characterization ofavailable methods would be necessary before an analytical methods-basedthreshold could be established.  Appropriate methods would need to be developedfor the detection of oat gluten.

**Finding 7.**  Theanalytical methods-based approach could be used to establish a threshold forgluten.  However, if this approach is used, the threshold should be replaced bya threshold established using one of the other approaches as quickly aspossible.

**b.  Safety Assessment-Based Approach.**  The safetyassessment-based approach could be used to establish a threshold for glutenbased on NOAELs or LOAELs reported in the literature in combination withappropriate uncertainty factors.  Clinical data in the literature are limited,but a few studies are available that meet the Threshold Working Group's dataquality criteria.  The currently available clinical studies do not reportNOAELs.  However, studies are available that could be used to establish a LOAELfrom which a threshold could be derived.

*Advantages*. Establishing a thresholdbased on NOAELs or LOAELs and the application of appropriate uncertaintyfactors to estimated exposure levels is fairly straightforward.  When there arelimited data in the literature, the application of appropriate uncertaintyfactors can provide confidence that the majority of the sensitive populationswill be protected.  Establishing thresholds using the safety assessment-basedapproach and currently available clinical data has the advantage of beingdirectly linked to biological effects.

*Limitations*. The primary limitation of thisapproach is the dearth of available clinical data and the general lack ofinformation about the impact of chronic low level consumption of gluten on theemergence of symptomatic disease in individuals with latent or silent celiacdisease.  At the current time, the size of the combined uncertainty factorsneeded would be substantial due to the general lack of data; applying largeuncertainty factors to the available data could lead to a gluten threshold thatis not achievable, as a practical matter, in foods.

We have identified several data gaps for gluten thatcontribute to current uncertainty about setting gluten thresholds.  The criticalareas of uncertainty and variability are:
- Intraspecies differences.  Safety assessments typically     apply a 10-fold uncertainty factor to account for the variability both     between individuals and variability in responses for a particular     individual.
- Chronic low-level exposure to gluten in     "gluten-free" diets:  Data, from either prospective studies or     long term clinical trials, are severely limited on the effect of a long     term gluten-free diet on disease symptoms.
- Adequacy of clinical trial data:  There is uncertainty as     to whether 4-week studies, or even 4 month studies, are of sufficient     duration to predict the consequences of long term ingestion of low levels     of gluten.  There is additional uncertainty as to whether currently available     clinical trials include the most sensitive individuals.  Accordingly,     there is uncertainty as to whether the standard 10-fold uncertainty factor     for variability within a species is sufficient to account for potential     highly sensitive individuals.  Additional uncertainty arises from the fact     that the published clinical trials were designed to identify LOAELs rather     than NOAELs. 
- Other.  Additional data gaps have been identified by the     Threshold Working Group; however, the working group concluded that uncertainties     associated with these factors were not sufficient to warrant additional     uncertainty factors.  These other data gaps include the following: (1) it     is uncertain what percentage of individuals with celiac disease are     sensitive to oat gluten and whether the levels to which they are sensitive     are equivalent to those observed for wheat; (2) variability in serving     sizes and related exposure factors; and (3) the effect of food processing     on the levels of gluten tolerated by individuals with celiac disease is     incompletely defined.  

The uncertainty associated with gluten thresholds arisesprimarily from the limited amount of clinical data.  The critical knowledge gapabout individuals with celiac disease is whether chronic, low-level exposure togluten in a gluten-free diet will cause any harm over a lifetime.  We are notaware of any prospective clinical trials that have examined the health ofindividuals with celiac disease on a gluten-free diet, and there is uncertaintyabout whether data from short term clinical trials will accurately predictsymptoms following chronic, low-level gluten exposure.  Conversely, thereappears to be only a small degree of uncertainty as to whether the mostsensitive celiac disease populations were included in the available clinicaltrials.

As discussed in Section III, there may be an oat-sensitivesubpopulation.  The possible existence of this oat sensitive subpopulationraises questions related to the definition of "gluten."  Becausethere are limited clinical data on the sensitivity of this subpopulation ofindividuals with celiac disease, the uncertainty related to the LOAELs orNOAELs for these individuals is high.  Nevertheless, it is unlikely that thesesindividuals are substantially more sensitive to oat gluten than they are towheat gluten.

Table IV-10 presents an example of how an overalluncertainty factor could be derived when estimating a threshold for glutenusing the safety assessment-based approach.  A standard uncertainty factor of 10should be applied for intraspecies differences in human responses to gluten.
Table IV-10. Example of UncertaintyFactors for the Safety-Assessment-Based Approach.DescriptionUncertainty FactorJustification  Intraspecies difference110
Standard for intraspecies  variability
  Extrapolation from LOAEL 210  Standard if NOAEL data not  available.  Supported by clinical trial data   Chronic, low level gluten  exposure36  Estimate using data from  gluten clinical trials. **Overall Uncertainty Factor4  = 600**
1 This includes both between- and within-individualvariability.

2 This includesboth a factor for converting the LOAEL to a NOAEL and an additional factor forthe uncertainty associated with that conversion factor.  Preliminary NOAEL datafrom an unpublished clinical trial (Fasano, 2005 personal communication)supports an approximate 10-fold difference between a NOAEL and published LOAELs(Catassi *et al*., 1993).

3 Estimated by comparing published LOAELs in an acute,single dose exposure (Ciclitira *et al*., 1984) with repeated exposureover four weeks (Catassi *et al*., 1993).

4 Uncertainty is likely to decrease as clinical trialdata becomes available.

**Finding 8.**  The safetyassessment-based approach is a viable approach to establish a threshold forgluten using currently available LOAEL data for celiac disease.  An overalluncertainty factor should be estimated from the data and applied to the LOAELto establish a threshold for gluten.  Any threshold derived from this approachshould be reevaluated as new research data become available.  Available dataare insufficient at the current time to use this approach to establish athreshold for oat gluten for those individuals with celiac disease who are alsosensitive to oats.  However, it is likely that a threshold based on wheat glutenwould be protective for individuals susceptible to oat gluten.

**c.  Risk Assessment-Based Approach.**  There are fewdata from human clinical trials that can be used to develop a dose-responsemodel for gluten and celiac disease.  In addition, limited data are availableon exposure; for example there are limited data on the actual levels of glutenin the diet of individuals on "gluten-free diets" and on the effects of lowlevel, chronic gluten exposure in individuals with silent or latent celiacdisease.  These limitations would lead to a very high level of uncertaintyassociated with models designed to predict the health effects of gluten in thediet.  Therefore, a scientifically defensible hazard characterization andexposure assessment are not possible at the current time.

**Finding 9.**  Use ofthe quantitative risk assessment-based approach to establish a threshold forgluten does not appear to be feasible at the present time.  However,considering the benefits that could be gained from using the risk assessment-basedapproach, priority should be given to establishing a research program toacquire the knowledge and data needed.

**d.  Statutorily-Derived Approach.**  The FALCPA doesnot include requirements or exemptions that could be used to establish a statutorily-derivedthreshold for gluten.  Also, the law does not define the term"gluten-free."  Potentially, a threshold could be established usingthe international standards currently under review by Codex (Codex AlimentariusCommision, 2004 **).**  However, the proposed Codex standards do not appearto be based on either a scientific rationale for a distinction betweennaturally gluten-free foods and foods processed to be free of gluten, or asystematic evaluation of clinical data related to the effect of gluten on acuteor chronic celiac disease etiology.  The levels being considered by Codex seemto be based on anecdotal evidence and on the levels of gluten that are presumedto be historically present in foods that have been called "gluten-free."

**Finding 10.**   Thereappear to be no suitable statutory requirements or exemptions that would serveas the rationale for using for a statutorily-derived approach to establish athreshold for gluten.  This approach is not viable.

Although the FALCPA requires establishing a definition forthe term "gluten-free" for food labeling, the quantity and quality of the dataneeded to accomplish this on a scientific basis are severely limited at thecurrent time relative to all three of the potentially viable approaches.  This wasaptly summarized by the consensus statement published after a conference ofexperts convened by the National Institutes of Health which noted that "Thestrict definition of a gluten-free diet remains controversial due to the lackof an accurate method to detect gluten in food products and the lack ofscientific evidence for what constitutes a safe amount of gluteningestion" (NIH, 2004).  These experts concluded that additional researchis needed to "Define the minimum safe exposure threshold of gluten in thediet relative to celiac disease" (NIH, 2004).

In view of the consensus opinion stated in the NIH report,the Threshold Working Group concluded that Finding 6 should be re-emphasized.Any approach used to establish a threshold for gluten to protect consumerswith, or susceptible to, celiac disease should be used in an iterative manner.Any such the threshold and approach should be re-examined periodically toconsider new scientific knowledge and data.
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AppendicesAppendix 1: Evaluation of Analytical Methods for Food AllergensAllergenManufactureraMethodValidationbSensitivity (LOD) (ppm)Quantitation (LOQ) (ppm)Raw and Processed Foods?Species SpecificityProtein(s) DetectedPracticalityPeanutAbkem IberiaPeanut DiagnoKitNo??Yes(For specified foods)??YesElisa SystemsPeanutNo??????NeogenAlert for Peanut No5 No???YesNeogenReveal for Peanut No5 No???YesNeogenVeratox for Peanut AOAC MLPT?2.5Yes(For specified foods)Peanut?YesR-BioPharmRiDASCREEN Peanut No2.5 ?Yes?Total YesR-BioPharmRIDASCREEN FAST Peanut AOAC MLPT1.5 2.5 Yes(For specified foods)PeanutTotalYesTecraPeanut Visual ImmunoassayNo0.5 2.5 Yes??YesTepnelBioKits PeanutAOAC MLPT0.1 1 Yes(For specified foods)PeanutConarachin (Ara h 1)YesTepnelBioKits Rapid Peanut No?No???YesMilkAbkem IberiaCasein DiagnoKitNo?0.16 Yes(For specified foods)?CaseinYesElisa SystemsBeta-lactoglogulinNo????Beta- Lactoglobulin?Elisa SystemsCaseinNo????Casein? NeogenAlert for Total MilkNo5 whole milk10 dry non-fat milkNoYes??YesNeogenVeratox for Total Milk No?2.5 Yes(For specified foods)??YesR-BioPharmRIDASCREEN Beta-LactoglobulinNo5 ???Beta-Lactoglobulin YesSafePathMilk ResidueNo????Beta-LactoglobulinYesTepnelBioKits BetaLG No7.5 25 Yes?Primarily Beta-LactoglobulinYesTepnelBioKits BSA No10 25Yes?Bovine Serum AlbuminYesTepnelBioKits Casein No1 2Yes?Primarily Alpha-CaseinYesEggElisa SystemsEggNo??????NeogenAlert for Egg No5 NYes(For specified foods)??YesNeogenVeratox for Egg No?2.5 Yes(For specified foods)??YesR-BioPharnRIDASCREEN Egg Protein No2 ?Yes(for specified foods ?)?WhiteYesSafePathEgg ResidueNo????OvomucoidYes TecraEgg Visual ImmunoassayNo0.5 0.6 Yes TotalYesTepnelBioKits Egg No0.1 0.5 Yes?OvomucoidYesTree NutsAbkem IberiaAlmond DiagnoKitNo?0.06 ???YesAbkem IberiaHazelnut DiagnoKitNo?0.08 ???YesElisa SystemAlmondNo??????Elisa SystemHazelnutNo??????NeogenAlert for Almond No5No???YesNeogenVeratox for Almond No?2.5???YesR-BioPharmRIDASCREEN Hazelnut No3.3 ???TotalYesR-BioPharmRIDASCREEN FAST Almond No1.7 2.5 ??TotalYesSoyElisa SystemSoyNo??????SafePathSoy ResiduesNo????Trypsin Inhibitor?TepnelSoya ProteinNo0.5% soy proteinin food sample0.5% soy proteinin food sample???YesCrustaceansAbkem IberiaCrustacean DiagnoKitNo?0.005?shrimp, crab,lobster and scampi.TropomyosinYesElisa SystemsCrustaceanNo????Tropomyosin?* Information from manufacturers web sites.+ MLPT - Multiple Laboratory Performance Tested
 
Appendix 2: Evaluation of Available Allergen Oral Challenge Studies.StudyPublishedTest PopulationFood Allergen TestedTest MaterialLowest Dose Tested(mg proteina)Dose ProgressionResponses at lowest dose tested?LOAEL Observed(mg proteina)Symptom(s) used to determine LOAELPopulation Dose/Response Data May, 1976YAsthmatic childrenPeanutRaw peanut25 2 -10 fold increaseY25Objective?MilkWhole?N?Objective?EggWhole?N?Objective?Dried?N250 +Objective?Bock *et al*., 1978YChildren with suspected allergyPeanutUnroasted???25ObjectiveNMilkDried nonfat???280ObjectiveNEggDried whole???1ObjectiveNSoyProtein isolate????ObjectiveNCashew?????ObjectiveNPecan?????ObjectiveNFilbert?????ObjectiveNCashew?????ObjectiveNPistaschio?????ObjectiveNPasterello *et al*., 1989YAdults with suspected allergyMilkDriedNot clear - differed for different foodsDose doubling?187ObjectiveNEgg whiteDriedN1500ObjectiveNHazelnutGroundN2775ObjectiveNBernhisel-Broadbent *et al*., 1992bYFish allergic children and adultsFishRaw and cooked extracts of 9 species?????NCaffarelli *et al*., 1995YInfants and children with no previous egg exposureEggDried egg0.042?N0.42ObjectiveNMagnolfi *et al*., 1996YAtopic childrenSoyFormula"1 drop" for infants6 defined dosesN360ObjectiveN88 mg soy protein for older children7 defined dosesY88ObjectiveNHourihane *et al.*, 1997aYPeanut allergic adultsPeanutPeanut flour0.0112 defined dosesN0.1SubjectiveY2ObjectiveHourihane *et al*., 1997bYPeanut allergic adultsPeanutWhole peanut? (Labial challenge)4 defined dosesY?ObjectiveNNelson *et al*., 1997YPeanut allergic adultsPeanutDefatted peanut0.4512 defined doses??SubjectiveNBellioni-Businco *et al*., 1999YMilk allergic childrenMilk"Fresh" whole milk"1 drop"?3.65 + Objective NHebling *et al*., 1999YFish allergic adultsFishCooked meat from 3 species of fish504 specified levelsY50Subjective and ObjectiveYZeiger *et al*., 1999YMilk allergic infants and childrenSoyFormula1 drop to 5 ml6 to 7 doublingsN52.2ObjectiveNOtolani *et al*., 2000YHazelnut allergic adultsHazelnutGround nuts 224 Dose doubling, possibly 4 levels???NSicherer *et al*., 2000YChildren with a variety of food allergiesbPeanut?400 or 500 mg of food6 or 7 specified levelsY??NMilk???NEgg???NSoy???NFish???NWheat???NEggesbo *et al*., 2001YChildrenEggPancakes260Dose doubling until reaction or maximum doseY260ObjectiveNBindslev-Jensen and Hansen in in Taylor *et al*., 2002N?FishCod???5mg of fish  Mackerel???500 mg of fishHerring???5 mg of fishPlaice???6000mg of fishBindslev-Jensen and Mortz in Taylor *et al*., 2002N?PeanutGround???40?NBindslev-Jensen and Norgaard in Taylor *et al*., 2002N?MilkWhole???180?NEggWhole raw???0.65?NBock in Taylor *et al*., 2002 BockN?PeanutGround???1.25?NMilkNonfat dried???67?NEggWhole or dried?????NFishMinced???200 mg of fish?NBurks and Christie in Taylor *et al*., 2002N?PeanutPeanut butter???100?NMilkNonfat dried???140?NEggWhole dried???200?NHill in Taylor *et al*., 2002N?PeanutPeanut butter???6?NMilkWhole???0.6?NEggRaw white???2?NHost in Taylor *et al*., 2002N?MilkFormula???75?NLack in Taylor *et al*., 2002N?PeanutGround???125?NMilkWhole???150?NEggCooked white???10?NRaw white???20?NMoneret-Vautrin #1 in Taylor *et al*., 2002N?PeanutGround???1.25 (single blind)2.5 (double blind)?NMilkWhole???30 (double blind150 (single blind)?NEggWhite???0.2?NFishMinced???15 mg of fish (single blind)65 mg of fish double blind?NMoneret-Vautrin #2 in Taylor *et al*., 2002N?PeanutGround???66?NEggWhite???26.5?N"National Jewish" in Taylor *et al*., 2002N?PeanutGround???2?NRance in Taylor *et al*., 2002N?PeanutGround???0.25?NMilkWhole???15?NEggWhole raw???0.13?NFishMinced???16 mg of fish?NZeiger in Taylor *et al*., 2002N?MilkFormula???1.5?NWensing *et al*., 2002aYHazelnut allergic adultsHazelnutRaw nuts17 specified dosesY1 Subjective and ObjectiveYWensing *et al*., 2002bYPeanut allergic adultsPeanutRoasted peanut meal0.03010 specified dosesN0.1Subjective and ObjectiveYFiocchi *et al*., 2003YChildren with allergy to both milk and soyMilkWhole43.24 specified dosesY43.2ObjectiveYSoyFormula21.821.8ObjectiveHansen *et al*., 2003YHazelnut allergic adultsHazelnutRaw and roasted nuts???32 - roasted16 - raw +Oral allergy syndromeNMorisset *et al*., 2003YUndefinedPeanutCrushed1.255 specified levelsY1.25ObjectiveNOil?N?ObjectiveNMilkLactose free0.36Y0.36ObjectiveNEggRaw white0.2Y0.2ObjectiveNSoyOil?N?ObjectiveNOsterballe and Bindslev-Jensen, 2003YEgg allergic childrenEggPasteurized whole egg2.98 specified dosesY2.9ObjectiveNPerry *et al.*, 2004Y?Milk, egg, peanut, soy, wheat??????N
Note: Question marks (?) in the table indicate either that the information was not given or could not be determined.a  Calculated based on the following estimate protein levels: 16% in raw hazelnuts, 20% in fish meat, 3.6%in whole milk, 37.5% in dried milk, 25% in whole peanut, 45% in defatted peanut flour, 10% in egg white, 84% in driedwhole egg, 26% in raw egg, 1.8% in soy formula (FAO, 1995; Wensing et al., 2002; Bindslev-Jensen et al., 2002).In studies involving fish, the amount of fish is given when there is insufficient information to calculate proteinlevels.b It is not clear if all children were tested with all allergens.

 
Appendix 3: Evaluation of Published Measurements of Protein Concentrations in Oils.Oil TypeReference aProtein Concentration(µg/g)Description of OilPublishedProtein Separation Method cProtein Quantitation MethodSoyTattrie and Yaguchi, 19730.96Refined, deodorizedYesChromatographyAmino Acid AnalysisKlurfeld and Kritchevsky, 19871.93CrudeYesAqueous ExtractionCommercial Bradford Assay0.72"Processed"Awazuhara *et al.*, 19980.0140.0170.0180.0230.0270.040Uncharacterized, commercialYesAqueous ExtractionLowery AssayReeves, 19990.0330.0420.0490.0570.0820.1140.222Fully refined, commercialNoUnknownAmino Acid AnalysisPaschke *et al*., 20010.03320.0353RefinedYesAcetone Precipitation Bradford Assay0.08980.10100.1380UnrefinedErrahali *et al*., 20020.32DeodorizedYesAqueous ExtractionUnknown1.80Cold pressedNordlee *et al*., 20020.16 - 20.8DegummedNoAqueous ExtractionAmino Acid Analysis0.043 - 6.8Refined0.033 - 3.1Bleached0.021 - 0.443DeodorizedPeanutKlurfeld and Kritchevsky, 19870.1200.1540.2040.2060.580ProcessedYesAqueous ExtractionBradford AssayHoffman and Collins-Williams, 19940.20.63.33.3Cold pressedYesAqueous ExtractionCommercial Coomassie Dye AssayTeuber *et al*., 19973.0 ± 0.35.7 ± 1.2Refined, bleached, deodorizedYesAqueous ExtractionCommercial Bradford Assay10.5 ± 0.410.7 ± 0.8UnrefinedOlszewski *et al*., 19980.100.130.150.160.20Refined, commercialYesAqueous ExtractionCommercial Bicinchoninic Acid (BCA) AssaySkinner and Haynes, 1998187CrudeNoAqueous ExtractionLowery and Commercial BCA Assay60Alkali refined, neutralized15Alkali refined, neutralized, bleached2.2Alkali refined, neutralized, bleached, deodorizedISEO, 19990.0470.0490.063Fully refined, commercialNoUnknownAmino Acid Analysis0.828Partially refined, commercialCrevel *et a*l., 2000b48Refined, neutralized, bleached, deodorizedNoAqueous ExtractionCommercial BCA Assay91220CrudePeeters *et al*., 20040.096.4Crude, noncommercialYesUnknownELISA (not described)2.55Cold pressedTree NutTeuber *et al.,* 1997 (Almond)2.2 ± 0.716.7 ± 0.8Refined, bleached, deodorizedYesAqueous ExtractionCommercial Bradford Assay12.7 ± 2.8Blend62.2 ± 2.2UnrefinedTeuber *et al*., 1997 (Walnut)7.0 ± 2.57.0 ± 0.89.2 ± 3.1Refined, bleached, deodorizedYesAqueous ExtractionCommercial Bradford Assay16.5 ± 2.4Unrefined20.4 ± 1.8BlendNote: Protein levels too low to detect or measure were reported by Tattrie and Yaguchi (1973),Hoffman and Collins-Williams (1994), Yeung and Collins (1996),  Peeters *et al*. (2004)for peanut oils and by Tattrie and Yaguchi (1973), Porras *et al*. (1985) for soy oils.These values were not included due to the lack of methodological information.aNone of the publications provide sufficient information to evaluate the overallextraction efficiency, accuracy, reproducibility, or precision of the method used. In addition,in most cases, it was not clear whether replicate samples were tested or whether replicatemeasurements were carried out for individual samples.bCrevel *et al*. (2000) is a review paper that includes previously unpublished data.These data are given here, but are considered unpublished because the research that generated thesevalues has not specifically been peer reviewed.
 
Appendix 4: Evaluation of Gluten Testing Methods.MethodaValidationSensitivity (LOD) (ppmb)Quantitation (LOQ)  (ppmb)Raw and Baked Foods?Species SpecificityProtein(s) DetectedPracticalityDiffchamb Transia Plate GlutenNo10??Wheat, triticale, rye, barley GliadinYesDiffchamb Transia Plate ProlaminsWorking Group on Prolamin Analysis and Toxicity3?YesWheat, triticale, rye, barleyGliadinYesIngensa Gluten EIANo3??Wheat, rye, barleyGliadin?Neogen Alert for GliadinNo20No?Wheat, rye, barleyGliadinYesNeogen Veratox for GliadinNo?5?Wheat, rye, barleyGliadinYesR-BioPharm RIDASCREEN GliadinProlamin Working Group Ring Studyc35YesWheat, rye, barleyGliadinsYesR-BioPharm RIDASCREEN FAST GliadinNo410YesWheat, rye, barley GliadinYesR-BioPharm RIDAQUICK GliadinNo5NoYesWheat, rye, barleyGliadinYesTepnel BioSystems Wheat GlutenAOAC160  2 - not validated  16YesWheat, triticale, ryeOmega gliadinYesTepnel BioSystems Gluten Rapid Test KitNo50 - breads, etc 200 - "highly processed flour"NoYesWheat, triticale, ryeOmega gliadinYesaInformation from manufacturers web sites:IngensaNeogenR-BioPharmTepnelbExpressed as ppm of gluten.cImmer *et al*., 2003.
 
Appendix 5: Evaluation of Gluten Oral Challenge StudiesStudyPublished Peer-reviewedPopulation TestedDiagnostic Assessment (Biomarker)Test Dose characterizedDose LevelDose DurationRaw Data AvailableFasano, 2005No; study is ongoing36 CD individualsIntestinal biopsy, symptoms 10 or 50 mg gluten/day3 months Catassi *et al*., 1993Yes10 CD children each doseIntestinal biopsy, symptoms100 mg or 500 mg gliadin/d200 mg or 1 gm gluten/d4 weeks Ciclitera *et al*., 1984Yes1 CD adults each doseIntestinal biopsy10 mg or 600 mg gliadin20 mg or 1.2gm gluten4 hr Ciclitera *et al*., 1984Yes7 CD adultsIntestinal biopsy, symptoms1.2 -2.4 mg gliadin/d2.8 -- 4.8mg gluten/d1 week Ciclitera *et al*., 1985Yes10 CD adultsIntestinal biopsy, symptoms1.2 -2.4 mg gliadin/d2.8 -- 4.8mg gluten/d6 weeks Montgomery *et al*, 1988Yes8 CD adultsIntestinal biopsy, symptoms, anti-gluten Ab 2.5 -- 5 gm gluten/d 6 months (median) Sturgess *et al*, 1994Yes4 CD adultsIntestinal biopsy 1 gm gliadin by intraduodenal infusion2 gm gluten6 hrs 
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