Food

Approaches to Establish Thresholds for Major Food Allergens and for Gluten in Food. Appendices

Table of Contents

Appendices

Appendix 1: Evaluation of Commercially Available Analytical Methods for Proteins from Major Food Allergen
Allergen Manufacturera Method Validationb Sensitivity (LODc) (ppm) Quantitation (LOQc) (ppm) Raw and Processed Foods? Species Specificity Protein(s) Detected Practicality
Peanut Abkem Iberia Peanut DiagnoKit No Not reported Not reported Yes (for specified foods) Not reported Not reported Yes
Elisa Systems Peanut JRC 0.5 1 Yes Peanut Ara h2 Yes
Elisa Systems Total Peanut No 0.5 1 Yes Peanut Total  
Neogen Alert for Peanut No 5 No Not reported Not reported Not reported Yes
Neogen Reveal for Peanut No 5 No Not reported Not reported Not reported Yes
Neogen Veratox for Peanut AOAC MLPT Not reported 2.5 Yes (for specified foods) Peanut Not reported Yes
R-BioPharm RiDASCREEN Peanut No 2.5 Not reported Yes Not reported Total Yes
R-BioPharm RIDASCREEN FAST Peanut AOAC MLPT 1.5 2.5 Yes (for specified foods) Peanut Total Yes
Tecra Peanut Visual Immunoassay No 0.5 2.5 Yes Not reported Not reported Yes
Tepnel BioKits Peanut AOAC MLPT 0.1 1 Yes (for specified foods) Peanut Conarachin (Ara h 1) Yes
Tepnel BioKits Rapid Peanut No Not reported No Not reported Not reported Not reported Yes
Milk
 
Abkem Iberia Casein DiagnoKit No Not reported 0.16 Yes (for specified foods) Not reported Casein Yes
Elisa Systems Beta-lactoglobulin No 0.5 1 Yes Not reported Beta- Lactoglobulin Yes
Elisa System Enhanced Beta-lactoglobulin No 1 1 Yes Not reported Beta-Lactoglobulin Yes
Elisa Systems Enhanced Casein No 1 1 Yes Not reported Casein Yes
Neogen Alert for Total Milk No 5 whole milk10 dry nonfat milk No Yes Not reported Not reported Yes
Neogen Veratox for Total Milk No Not reported 2.5 Yes (for specified foods) Not reported Not reported Yes
R-BioPharm RIDASCREEN Beta-Lactoglobulin No 0.2 5 Not reported Not reported Beta- Lactoglobulin Yes
SafePath Milk Residue No Not reported Not reported Not reported Not reported Beta- Lactoglobulin Yes
Tepnel BioKits BLG No 7.5 25 Yes Not reported Primarily Beta-Lactoglobulin Yes
Tepnel BioKits BSA No 10 25 Yes Not reported Bovine Serum Albumin Yes
Tepnel BioKits Casein No 1 2 Yes Not reported Primarily Alpha-Casein Yes
Tepnel BioKits Casein Rapid No Not reported Not reported Yes (for specified foods) Not reported Casein Yes
Egg Elisa Systems Egg No 0.5 1 Yes Ovalbumin, Ovamucoid Yes Not reported
Neogen Alert for Egg No 5 N Yes (for specified foods) Not reported Not reported Yes
Neogen Veratox for Egg No Not reported 2.5 Yes (for specified foods) Not reported Not reported Yes
R-BioPharn RIDASCREEN Egg Protein No 2 Not reported Yes (for specified foods) Not reported White Yes
SafePath Egg Residue No Not reported Not reported Not reported Not reported Ovomucoid Yes
Tecra Egg Visual Immunoassay No 0.5 0.6 Yes   Total Yes
Tepnel BioKits Egg No 0.1 0.5 Yes Not reported Ovomucoid Yes
Tree Nuts Abkem Iberia Almond DiagnoKit No Not reported 0.06 Not reported Not reported Not reported Yes
Abkem Iberia Hazelnut DiagnoKit No Not reported 0.08 Not reported Not reported Not reported Yes
Elisa System Almond No 0.5 1 Yes Not reported Not reported Yes
Elisa System Hazelnut No 0.25 0.5 Yes Not reported Not reported Yes
Neogen Alert for Almond No 5 No Not reported Not reported Not reported Yes
Neogen Veratox for Almond No Not reported 2.5 Not reported Not reported Not reported Yes
R-BioPharm RIDASCREEN Hazelnut No 3.3 Not reported Not reported Not reported Total Yes
R-BioPharm RIDASCREEN FAST Almond No 1.7 2.5 Not reported Not reported Total Yes
Soy Elisa System Soy No 1 1 Yes (for specified foods) Not reported Trypsin Inhibitor Yes
Elisa Systems Enhanced Soy No 1 2.5 Yes (for specified foods) Not reported Soy flour proteins Yes
SafePath Soy Residues No Not reported Not reported Not reported Not reported Trypsin Inhibitor Not reported
Neogen Alert for Soy Flour No 5 Not reported Yes Not reported Not reported Yes
Neogen Veratox for Soy Flour No 2.5 2.5 Yes Not reported Not reported Yes
Tepnel Soya Protein No 0.5% soy protein in food sample 0.5% soy protein in food sample Not reported Not reported Not reported Yes
Crustaceans Abkem Iberia Crustacean DiagnoKit No Not reported 0.005 Not reported shrimp, crab, lobster and scampi. Tropomyosin Yes
Elisa Systems Crustacean (18 species) No 0.05 0.05 Yes (for specified foods) Not reported Tropomyosin Yes
Fish No commercial methods are available.                
Wheat See Appendix 4 for gluten methods. No other commercial methods are available.                

a Information from manufacturers web sites, except for information on the Elisa System Crustacean test kit from FDA Docket #2005N-0231, comment number EC1.

bMLPT - Multiple Laboratory Performance Tested; JRC- European Commission Joint Research Centre; AOAC = AOAC International.

cLOD = Limit of detection, LOQ = Limit of quantiation.

 Appendix 2: Evaluation of Available Allergen Oral Challenge Studies.
Study Published Test Population Food Allergen Tested Test Material Lowest Dose Tested (mg proteina) Dose Progression Responses at lowest dose tested? LOAEL Observed (mg proteina, b) Sign(s)s or symptom(s) used to determine LOAEL Population Dose/Response Data
May, 1976 Yes 38 asthmatic children, 8 reacted to peanut, 1 to milk, 4 t o egg Peanut, Raw peanut 25 2-10 fold increase Yes 25 Objective Not reported
Milk Whole Not reported 2-10 fold increase No Not reported Objective Not reported
Egg Whole Not reported 2-10 fold increase No Not reported Objective Not reported
Eggs Dried Not reported 2-10 fold increase No 250 Objective Not reported
Bock et al., 1978 Yes 68 children with suspected allergy, 12 reacted to peanut, 10 to milk, 10 to egg, 5 to soy, 2 to cashew, 1 each to pecan, filbert, pistachio Peanut Unroasted Not reported Not reported Not reported 25 Objective No
Milk Dried nonfat Not reported Not reported Not reported 280 Objective No
Egg Dried whole Not reported Not reported Not reported 1 Objective No
Soy Protein isolate Not reported Not reported Not reported Not reported Objective No
Cashew Not reported Not reported Not reported Not reported Not reported Objective No
Pecan Not reported Not reported Not reported Not reported Not reported Objective No
Filbert Not reported Not reported Not reported Not reported Not reported Objective No
Cashew Not reported Not reported Not reported Not reported Not reported Objective No
Pistaschio Not reported Not reported Not reported Not reported Not reported Objective No
Pasterello et al., 1989 Yes 23 adults with suspected allergy, 4 reacted t milk, 2 to hazelnut, and 1 each to egg and wheat Milk Dried Not clear - differed for different foods Dose doubling Not reported 187 Objective No
Egg white Dried   No 1500 Objective No
Hazelnut Ground     No 2775 Objective No
Bernhisel-Broadbent et al., 1992b Yes 11 fish allergic children and adults Fish Raw and cooked extracts of 9 species Not reported Not reported Not reported Not reported Not reported No
Caffarelli et al., 1995 Yes 21 infants and children with no previous egg exposure, 14 reacted Egg Dried egg 0.042 Not reported No 0.42 Objective No
Magnolfi et al., 1996 Yes 131 skin prick positive children, 8 reacted Soy Formula "1 drop" for infants 6 defined doses No 360 Objective No
88 mg soy protein for older children 7 defined doses Yes 88 Objective No
Hourihane et al., 1997a Yes 14 peanut allergic adults, 8 reacted Peanut Peanut flour 0.01 12 defined doses No

0.1

2

Subjective

Objective

Yes
Hourihane et al., 1997b Yes 60 peanut allergic adults Peanut Whole peanut ? (Labial challenge) 4 defined doses Yes Not reported Objective No
Nelson et al., 1997 Yes 12 peanut allergic adults Peanut Defatted peanut 0.45 12 defined doses Not reported Not reported Subjective No
Bellioni-Businco et al., 1999 Yes 26 milk allergic children Milk "Fresh" whole milk "1 drop" Not reported 3.65   Objective No
Hebling et al., 1999 Yes 9 fish allergic adults Fish Cooked meat from 3 species of fish 50 4 specified levels Yes 50 Subjective and Objective Yes
Zeiger et al., 1999 Yes 93 milk allergic infants and children, 13 reacted to soy Soy Formula 1 drop to 5 ml 6 to 7 doublings No 522 Objective No
Otolani et al., 2000 Yes 86 hazelnut allergic adults Hazelnut Ground nuts 224 Dose doubling, possibly 4 levels Not reported Not reported Not reported No
Sicherer et al., 2000 Yes 196 children with a variety of food allergiesc Peanut Not reported 400 or 500 mg of food 6 or 7 specified levels Yes Not reported Not reported No
Milk Not reported Not reported Not reported No
Egg Not reported Not reported Not reported No
Soy Not reported Not reported Not reported No
Fish Not reported Not reported Not reported No
Wheat Not reported Not reported Not reported No
Eggesbo et al., 2001 Yes 41 children with reported allergy, 5 tested by DBPCFC Egg Pancakes 260 Dose doubling until reaction or maximum dose Yes 260 Objective No
Bindslev-Jensen and Hansen in Taylor et al., 2002 No 14 patients, not clear whether all were challenged with each fish Fish Cod Not reported Not reported   5 mg of fish    
Mackerel Not reported Not reported Not reported 500mg of fish    
Herring Not reported Not reported Not reported 5mg of fish    
Plaice Not reported Not reported Not reported 6000mg of fish    
Bindslev-Jensen and Mortz in Taylor et al., 2002 No 5 patients Peanut Ground Not reported Not reported Not reported 40 Not reported No
Bindslev-Jensen and Norgaard in Taylor et al., 2002 No 3 milk allergic, 7 egg allergic patients Milk Whole Not reported Not reported Not reported 180 Not reported No
Egg Whole raw Not reported Not reported Not reported 0.65 Not reported No
Bock in Taylor et al., 2002 No 69 peanut allergic, 66 milk allergic, 91 egg allergic, 8 fish allergic patients Peanut Ground Not reported Not reported Not reported 1.25 Not reported No
Milk Nonfat dried Not reported Not reported Not reported 67 Not reported No
Egg Whole or dried Not reported Not reported Not reported Not reported Not reported No
Fish Minced Not reported Not reported Not reported 200 mg of fish Not reported No
Burks and Christie in Taylor et al., 2002 No 10 peanut allergic, 21 milk allergic, 25 egg allergic patients Peanut Peanut butter Not reported Not reported Not reported 100 Not reported No
Milk Nonfat dried Not reported Not reported Not reported 140 Not reported No
Egg Whole dried Not reported Not reported Not reported 200 Not reported No
Hill in Taylor et al., 2002 No 100 patients each for peanut, milk, egg Peanut Peanut butter Not reported Not reported Not reported 6 Not reported No
Milk Whole Not reported Not reported Not reported 0.6 Not reported No
Egg Raw white Not reported Not reported Not reported 2 Not reported No
Host in Taylor et al., 2002 No 15 milk allergic patients Milk Forumula Not reported Not reported Not reported 75 Not reported No
Lack in Taylor et al., 2002 No 6 peanut, 6 milk, 18 egg allergic patients Peanut Ground Not reported Not reported Not reported 125 Not reported No
Milk Whole Not reported Not reported Not reported 150 Not reported No
Egg Cooked white Not reported Not reported Not reported 10 Not reported No
Raw white Not reported Not reported Not reported 20 Not reported No
Moneret-Vautrin #1 in Taylor et al., 2002 No 28 peanut allergic, 6 milk allergic, 19 egg allergic, 4 fish allergic patients Peanut Ground Not reported Not reported Not reported 1.25 (single blind)2.5 (double blind) Not reported No
Milk Whole Not reported Not reported Not reported 30 (double blind)150 (single blind)0.2 Not reported No
Egg White Not reported Not reported Not reported 15mg of fish (single blind) Not reported No
Fish Minced Not reported Not reported Not reported 65mg of fish (double blind) Not reported No
Moneret-Vautrin #2 in Taylor et al., 2002 No 9 peanut allergic, 8 egg allergic patients Peanut Ground Not reported Not reported Not reported 66 Not reported No
Egg White Not reported Not reported Not reported 26.5 Not reported No
"National Jewish" in Taylor et al., 2002 No 3 peanut allergic patients Peanut Ground Not reported Not reported Not reported 2 Not reported No
Rance in Taylor et al., 2002 No 74 peanut allergic, 31 milk allergic, 38 egg allergic, 6 fish allergic patients Peanut Ground Not reported Not reported Not reported 0.25 Not reported No
Milk Whole Not reported Not reported Not reported 15 Not reported No
Egg Whole raw Not reported Not reported Not reported 0.13 Not reported No
Fish Minced Not reported Not reported Not reported 16mg of fish Not reported No
Zeiger in Taylor et al., 2002 No 56 milk allergic patients Milk Formula Not reported Not reported Not reported 1.5 Not reported No
Wensing et al., 2002a Yes 31 hazelnut allergic adults Hazelnut Raw nuts 1 7 specified doses Yes 1 Subjective and Objective Yes
Wensing et al., 2002b Yes 26 peanut allergic adults Peanut Roasted peanut meal 0.030 10 specified doses No 0.1 Subjective and Objective Yes
Fiocchi et al., 2003 Yes 18 children with allergy to both milk and soy Milk Whole 43.2 4 specified doses Yes 43.2 Objective Yes
    Soy Formula 21.8     21.8 Objective  
Hansen et al., 2003 Yes 17 hazelnut allergic adults Hazelnut Raw and roasted nuts Not reported Not reported Not reported 32 - roasted16 - raw Oral allergy syndrome No
Morisset et al., 2003 Yes Undefined Peanut Crushed 1.25 5 specified levels Yes 1.25 Objective No
  Oil Not reported No Not reported Objective No
Milk Lactose free 0.36 Yes 0.36 Objective No
Egg Raw white 0.2 Yes 0.2 Objective No
Soy Oil Not reported No Not reported Objective No
Osterballe and Bindslev-Jensen, 2003 Yes 56 egg allergic children Egg Pasteurized whole egg 2.9 8 specified doses Yes 2.9 Objective No
Perry et al., 2004 Yes Not reported Milk, egg, peanut, soy, wheat Not reported Not reported Not reported Not reported Not reported Not reported No
Scibilia et al., 2006 Yes 27 wheat allergic adults Wheat Flour, raw and cooked 15 7 specified doses Yes 15 Objective Yes

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 When responses are observed at the lowest dose tested, the reported LOAEL may not represent the lowest dose at which a reaction could occur.

c It is not clear if all children were tested with all allergens.

Appendix 3: Evaluation of Published Measurements of Protein Concentrations in Oils.
Oil Type Reference a Protein Concentration (ug/g) Description of Oil Published Protein Separation Method Protein Quantitation Method
Soy Tattrie and Yaguchi, 1973 0.96 Refined, deodorized

Yes

Chromatography Amino Acid Analysis
Klurfeld and Kritchevsky, 1987

1.93

0.72

Crude

"Processed"

Yes

Aqueous Extraction Commercial Bradford Assay
Awazuhara et al., 1998

0.014

0.017

0.018

0.023

0.027

0.040

Uncharacterized, commercial

Yes

Aqueous Extraction Lowery Assay
Reeves, 1999

0.033

0.042

0.049

0.057

0.082

0.114

0.222

Fully refined, commercial

No

Unknown Amino Acid Analysis
Paschke et al., 2001

0.0332

0.0353

0.0898

0.1010

0.1380

Refined

Unrefined

Yes

Acetone Precipitation Bradford Assay
Errahali et al., 2002

0.32

1.80

Deodorized

Cold pressed

Yes

Aqueous Extraction Unknown
Nordlee et al., 2002

0.16 - 20.8

0.043 - 6.8

0.033 - 3.1

0.021 - 0.443

Degummed

Refined

Bleached

Deodorized

No

Aqueous Extraction Amino Acid Analysis
Peanut Klurfeld and Kritchevsky, 1987

0.120

0.154

0.204

0.206

0.580

Processed

Yes

Aqueous Extraction Bradford Assay
Hoffman and Collins-Williams, 1994

0.2

0.6

3.3

3.3

Cold pressed

Yes

Aqueous Extraction Commercial Coomassie Dye Assay
Teuber et al., 1997

3.0 ± 0.3

5.7 ± 1.2

10.5 ± 0.4

10.7 ± 0.8

Refined, bleached, deodorized

Unrefined

Yes

Aqueous Extraction Commercial Bradford Assay
Olszewski et al., 1998

0.10

0.13

0.15

0.16

0.20

Refined, commercial

Yes

Aqueous Extraction Commercial Bicinchoninic Acid (BCA) Assay
Skinner and Haynes, 1998 187 Crude

No

Aqueous Extraction Lowery and Commercial BCA Assay
60 Alkali refined, neutralized      
15 Alkali refined, neutralized, bleached      
2.2 Alkali refined, neutralized, bleached, deodorized      
ISEO, 1999

0.047

0.049

0.063

Fully refined, commercial

No

Unknown Amino Acid Analysis
0.828 Partially refined, commercial      
Crevel et al., 2000 b

48

91

Refined, neutralized, bleached, deodorized

No

Aqueous Extraction Commercial BCA Assay
220 Crude      
Peeters et al., 2004

0.09

6.4

Crude, noncommercial

Yes

Unknown ELISA (not described)
2.55 Cold pressed      
Tree Nut Teuber et al., 1997 (Almond)

2.2 ± 0.7

16.7 ± 0.8

Refined, bleached, deodorized

Yes

Aqueous Extraction Commercial Bradford Assay
12.7 ± 2.8 Blend      
62.2 ± 2.2 Unrefined      
Teuber et al., 1997 (Walnut)

7.0 ± 2.5

7.0 ± 0.8

9.2 ± 3.1

Refined, bleached, deodorized

Yes

Aqueous Extraction Commercial Bradford Assay
16.5 ± 2.4 Unrefined      
20.4 ± 1.8 Blend      

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.

a None 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.

b Crevel 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.
Method a Validation Sensitivity (LOD) (ppm gluten) Quantitation (LOQ) (ppm gluten) Raw and Baked Foods? Species Specificity Protein(s) Detected Practicality
Diffchamb Transia Plate Gluten No 10 Not reported Not reported Wheat, triticale, rye, barley Gliadin Yes
Diffchamb Transia Plate Prolamins Working Group on Prolamin Analysis and Toxicity 3 Not reported Yes Wheat, triticale, rye, barley Gliadin Yes
Ingensa Gluten EIA No 3 Not reported Not reported Wheat, rye, barley Gliadin Not reported
Neogen Alert for Gliadin No 10 No Not reported Wheat, rye, barley Gliadin Yes
Neogen Veratox for Gliadin No 5 5 Not reported Wheat, rye, barley Gliadin Yes
R-BioPharm RIDASCREEN Gliadin Prolamin Working Group Ring Study b 3 5 Yes Wheat, rye, barley Gliadin Yes
R-BioPharm RIDASCREEN FAST Gliadin No 10 10 Yes Wheat, rye, barley Gliadin Yes
R-BioPharm RIDAQUICK Gliadin No 5 No Yes Wheat, rye, barley Gliadin Yes
Tepnel BioSystems Wheat Gluten AOAC

160

2 - not validated

16 Yes Wheat, triticale, rye Omega gliadin Yes
Tepnel BioSystems Gluten Rapid Test Kit No

50 - breads, etc

200 - "highly processed flour

No Yes Wheat, triticale, rye Omega gliadin Yes

a Information from manufacturers web sites:

Ingensa

Neogen Food Allergen Test Kits

R-BioPharm Food and Feed Analysis RIDASCREEN® Gliadin

Tepnel BioSystems

b Immer et al., 2003.

Appendix 5: Evaluation of Gluten Oral Challenge Studies.
Study Published Test Population Test Material Dose Level(s) Duration Diagnostic Assessment (Biomarker) Individual Response Data?

Fasano, 2005;

Abstract: Catassi et al., 2005b

No; analysis is ongoing 33 of 46 adults completed study Not reported 0, 10 or 50 mg gluten/day 3 months Intestinal biopsy, symptoms Not reported
Catassi et al., 1993 Yes 20 children (10 each dose level) Commercial crude gliadin 100 mg or 500 mg gliadin/day 4 weeks Intestinal biopsy, symptoms Yes
Ciclitera et al., 1984 Yes 1 adult White flour milled from Kolibri strain of wheat 10, 600, and 1000 mg gliadin Intraduodenal infusion over 2 hr period; 3 doses on separate days Intestinal biopsy Yes
Ciclitera et al., 1984 Yes 3 adults White flour milled from Kolibri strain of wheat 1000 mg of 4 gliadin subfractions Intraduodenal infusion each subfraction at variable intervals of 3 to 11 days Intestinal biopsy, symptoms Yes
Ciclitera et al., 1985 Yes 10 adults Not reported; article states that gluten-free bread usually contains 0.2 to 0.4 mg gliadin/30-g slice 6 slices/day Juvela gluten-free bread; oral 6 weeks Intestinal biopsy,symptoms Yes
Montgomery et al, 1988 Yes

12 adults on strict gluten-free diet and

13 adults on low-gluten diet

Not reported

Gluten-free diet

Low-gluten diet (2.5 to 5 gm gluten/day)

Gluten-free diet: 6 to 27 months (mean 13 months);

Low-gluten diet: 3 to 14 months (mean 6 months)

Intestinal biopsy, symptoms, anti-gluten Ab Yes, graphs
Sturgess et al, 1994 Yes 4 adults Undigested gliadin prepared from Kolibri wheat flour by standard method; oligopeptides synthesized and analyzed 1 gm gliadin or 200 mg of synthetic peptides/dose 2 hours by infusion Intestinal biopsy hourly for 6 hrs after start infusion Yes; as percentage enteropathic change

June 2005 Draft Report: Approaches to Establish Thresholds for Major Food Allergens and for Gluten in Food

March 2006 FDA's Responses to Public Comments on the Draft Report

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