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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
AllergenManufactureraMethodValidationbSensitivity (LODc) (ppm)Quantitation (LOQc) (ppm)Raw and Processed Foods?Species SpecificityProtein(s) DetectedPracticality
PeanutAbkem IberiaPeanut DiagnoKitNoNot reportedNot reportedYes (for specified foods)Not reportedNot reportedYes
Elisa SystemsPeanutJRC0.51YesPeanutAra h2Yes
Elisa SystemsTotal PeanutNo0.51YesPeanutTotal 
NeogenAlert for PeanutNo5NoNot reportedNot reportedNot reportedYes
NeogenReveal for PeanutNo5NoNot reportedNot reportedNot reportedYes
NeogenVeratox for PeanutAOAC MLPTNot reported2.5Yes (for specified foods)PeanutNot reportedYes
R-BioPharmRiDASCREEN PeanutNo2.5Not reportedYesNot reportedTotalYes
R-BioPharmRIDASCREEN FAST PeanutAOAC MLPT1.52.5Yes (for specified foods)PeanutTotalYes
TecraPeanut Visual ImmunoassayNo0.52.5YesNot reportedNot reportedYes
TepnelBioKits PeanutAOAC MLPT0.11Yes (for specified foods)PeanutConarachin (Ara h 1)Yes
TepnelBioKits Rapid PeanutNoNot reportedNoNot reportedNot reportedNot reportedYes
Milk
 
Abkem IberiaCasein DiagnoKitNoNot reported0.16Yes (for specified foods)Not reportedCaseinYes
Elisa SystemsBeta-lactoglobulinNo0.51YesNot reportedBeta- LactoglobulinYes
Elisa SystemEnhanced Beta-lactoglobulinNo11YesNot reportedBeta-LactoglobulinYes
Elisa SystemsEnhanced CaseinNo11YesNot reportedCaseinYes
NeogenAlert for Total MilkNo5 whole milk10 dry nonfat milkNoYesNot reportedNot reportedYes
NeogenVeratox for Total MilkNoNot reported2.5Yes (for specified foods)Not reportedNot reportedYes
R-BioPharmRIDASCREEN Beta-LactoglobulinNo0.25Not reportedNot reportedBeta- LactoglobulinYes
SafePathMilk ResidueNoNot reportedNot reportedNot reportedNot reportedBeta- LactoglobulinYes
TepnelBioKits BLGNo7.525YesNot reportedPrimarily Beta-LactoglobulinYes
TepnelBioKits BSANo1025YesNot reportedBovine Serum AlbuminYes
TepnelBioKits CaseinNo12YesNot reportedPrimarily Alpha-CaseinYes
TepnelBioKits Casein RapidNoNot reportedNot reportedYes (for specified foods)Not reportedCaseinYes
EggElisa SystemsEggNo0.51YesOvalbumin, OvamucoidYesNot reported
NeogenAlert for EggNo5NYes (for specified foods)Not reportedNot reportedYes
NeogenVeratox for EggNoNot reported2.5Yes (for specified foods)Not reportedNot reportedYes
R-BioPharnRIDASCREEN Egg ProteinNo2Not reportedYes (for specified foods)Not reportedWhiteYes
SafePathEgg ResidueNoNot reportedNot reportedNot reportedNot reportedOvomucoidYes
TecraEgg Visual ImmunoassayNo0.50.6Yes TotalYes
TepnelBioKits EggNo0.10.5YesNot reportedOvomucoidYes
Tree NutsAbkem IberiaAlmond DiagnoKitNoNot reported0.06Not reportedNot reportedNot reportedYes
Abkem IberiaHazelnut DiagnoKitNoNot reported0.08Not reportedNot reportedNot reportedYes
Elisa SystemAlmondNo0.51YesNot reportedNot reportedYes
Elisa SystemHazelnutNo0.250.5YesNot reportedNot reportedYes
NeogenAlert for AlmondNo5NoNot reportedNot reportedNot reportedYes
NeogenVeratox for AlmondNoNot reported2.5Not reportedNot reportedNot reportedYes
R-BioPharmRIDASCREEN HazelnutNo3.3Not reportedNot reportedNot reportedTotalYes
R-BioPharmRIDASCREEN FAST AlmondNo1.72.5Not reportedNot reportedTotalYes
SoyElisa SystemSoyNo11Yes (for specified foods)Not reportedTrypsin InhibitorYes
Elisa SystemsEnhanced SoyNo12.5Yes (for specified foods)Not reportedSoy flour proteinsYes
SafePathSoy ResiduesNoNot reportedNot reportedNot reportedNot reportedTrypsin InhibitorNot reported
NeogenAlert for Soy FlourNo5Not reportedYesNot reportedNot reportedYes
NeogenVeratox for Soy FlourNo2.52.5YesNot reportedNot reportedYes
TepnelSoya ProteinNo0.5% soy protein in food sample0.5% soy protein in food sampleNot reportedNot reportedNot reportedYes
CrustaceansAbkem IberiaCrustacean DiagnoKitNoNot reported0.005Not reportedshrimp, crab, lobster and scampi.TropomyosinYes
Elisa SystemsCrustacean (18 species)No0.050.05Yes (for specified foods)Not reportedTropomyosinYes
FishNo commercial methods are available.        
WheatSee 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.
StudyPublishedTest PopulationFood Allergen TestedTest MaterialLowest Dose Tested (mg proteina)Dose ProgressionResponses at lowest dose tested?LOAEL Observed (mg proteina, b)Sign(s)s or symptom(s) used to determine LOAELPopulation Dose/Response Data
May, 1976Yes38 asthmatic children, 8 reacted to peanut, 1 to milk, 4 t o eggPeanut,Raw peanut252-10 fold increaseYes25ObjectiveNot reported
MilkWholeNot reported2-10 fold increaseNoNot reportedObjectiveNot reported
EggWholeNot reported2-10 fold increaseNoNot reportedObjectiveNot reported
EggsDriedNot reported2-10 fold increaseNo250ObjectiveNot reported
Bock et al., 1978Yes68 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, pistachioPeanutUnroastedNot reportedNot reportedNot reported25ObjectiveNo
MilkDried nonfatNot reportedNot reportedNot reported280ObjectiveNo
EggDried wholeNot reportedNot reportedNot reported1ObjectiveNo
SoyProtein isolateNot reportedNot reportedNot reportedNot reportedObjectiveNo
CashewNot reportedNot reportedNot reportedNot reportedNot reportedObjectiveNo
PecanNot reportedNot reportedNot reportedNot reportedNot reportedObjectiveNo
FilbertNot reportedNot reportedNot reportedNot reportedNot reportedObjectiveNo
CashewNot reportedNot reportedNot reportedNot reportedNot reportedObjectiveNo
PistaschioNot reportedNot reportedNot reportedNot reportedNot reportedObjectiveNo
Pasterello et al., 1989Yes23 adults with suspected allergy, 4 reacted t milk, 2 to hazelnut, and 1 each to egg and wheatMilkDriedNot clear - differed for different foodsDose doublingNot reported187ObjectiveNo
Egg whiteDried No1500ObjectiveNo
HazelnutGround  No2775ObjectiveNo
Bernhisel-Broadbent et al., 1992bYes11 fish allergic children and adultsFishRaw and cooked extracts of 9 speciesNot reportedNot reportedNot reportedNot reportedNot reportedNo
Caffarelli et al., 1995Yes21 infants and children with no previous egg exposure, 14 reactedEggDried egg0.042Not reportedNo0.42ObjectiveNo
Magnolfi et al., 1996Yes131 skin prick positive children, 8 reactedSoyFormula"1 drop" for infants6 defined dosesNo360ObjectiveNo
88 mg soy protein for older children7 defined dosesYes88ObjectiveNo
Hourihane et al., 1997aYes14 peanut allergic adults, 8 reactedPeanutPeanut flour0.0112 defined dosesNo

0.1

2

Subjective

Objective

Yes
Hourihane et al., 1997bYes60 peanut allergic adultsPeanutWhole peanut? (Labial challenge)4 defined dosesYesNot reportedObjectiveNo
Nelson et al., 1997Yes12 peanut allergic adultsPeanutDefatted peanut0.4512 defined dosesNot reportedNot reportedSubjectiveNo
Bellioni-Businco et al., 1999Yes26 milk allergic childrenMilk"Fresh" whole milk"1 drop"Not reported3.65 ObjectiveNo
Hebling et al., 1999Yes9 fish allergic adultsFishCooked meat from 3 species of fish504 specified levelsYes50Subjective and ObjectiveYes
Zeiger et al., 1999Yes93 milk allergic infants and children, 13 reacted to soySoyFormula1 drop to 5 ml6 to 7 doublingsNo522ObjectiveNo
Otolani et al., 2000Yes86 hazelnut allergic adultsHazelnutGround nuts224Dose doubling, possibly 4 levelsNot reportedNot reportedNot reportedNo
Sicherer et al., 2000Yes196 children with a variety of food allergiescPeanutNot reported400 or 500 mg of food6 or 7 specified levelsYesNot reportedNot reportedNo
MilkNot reportedNot reportedNot reportedNo
EggNot reportedNot reportedNot reportedNo
SoyNot reportedNot reportedNot reportedNo
FishNot reportedNot reportedNot reportedNo
WheatNot reportedNot reportedNot reportedNo
Eggesbo et al., 2001Yes41 children with reported allergy, 5 tested by DBPCFCEggPancakes260Dose doubling until reaction or maximum doseYes260ObjectiveNo
Bindslev-Jensen and Hansen in Taylor et al., 2002No14 patients, not clear whether all were challenged with each fishFishCodNot reportedNot reported 5 mg of fish  
MackerelNot reportedNot reportedNot reported500mg of fish  
HerringNot reportedNot reportedNot reported5mg of fish  
PlaiceNot reportedNot reportedNot reported6000mg of fish  
Bindslev-Jensen and Mortz in Taylor et al., 2002No5 patientsPeanutGroundNot reportedNot reportedNot reported40Not reportedNo
Bindslev-Jensen and Norgaard in Taylor et al., 2002No3 milk allergic, 7 egg allergic patientsMilkWholeNot reportedNot reportedNot reported180Not reportedNo
EggWhole rawNot reportedNot reportedNot reported0.65Not reportedNo
Bock in Taylor et al., 2002No69 peanut allergic, 66 milk allergic, 91 egg allergic, 8 fish allergic patientsPeanutGroundNot reportedNot reportedNot reported1.25Not reportedNo
MilkNonfat driedNot reportedNot reportedNot reported67Not reportedNo
EggWhole or driedNot reportedNot reportedNot reportedNot reportedNot reportedNo
FishMincedNot reportedNot reportedNot reported200 mg of fishNot reportedNo
Burks and Christie in Taylor et al., 2002No10 peanut allergic, 21 milk allergic, 25 egg allergic patientsPeanutPeanut butterNot reportedNot reportedNot reported100Not reportedNo
MilkNonfat driedNot reportedNot reportedNot reported140Not reportedNo
EggWhole driedNot reportedNot reportedNot reported200Not reportedNo
Hill in Taylor et al., 2002No100 patients each for peanut, milk, eggPeanutPeanut butterNot reportedNot reportedNot reported6Not reportedNo
MilkWholeNot reportedNot reportedNot reported0.6Not reportedNo
EggRaw whiteNot reportedNot reportedNot reported2Not reportedNo
Host in Taylor et al., 2002No15 milk allergic patientsMilkForumulaNot reportedNot reportedNot reported75Not reportedNo
Lack in Taylor et al., 2002No6 peanut, 6 milk, 18 egg allergic patientsPeanutGroundNot reportedNot reportedNot reported125Not reportedNo
MilkWholeNot reportedNot reportedNot reported150Not reportedNo
EggCooked whiteNot reportedNot reportedNot reported10Not reportedNo
Raw whiteNot reportedNot reportedNot reported20Not reportedNo
Moneret-Vautrin #1 in Taylor et al., 2002No28 peanut allergic, 6 milk allergic, 19 egg allergic, 4 fish allergic patientsPeanutGroundNot reportedNot reportedNot reported1.25 (single blind)2.5 (double blind)Not reportedNo
MilkWholeNot reportedNot reportedNot reported30 (double blind)150 (single blind)0.2Not reportedNo
EggWhiteNot reportedNot reportedNot reported15mg of fish (single blind)Not reportedNo
FishMincedNot reportedNot reportedNot reported65mg of fish (double blind)Not reportedNo
Moneret-Vautrin #2 in Taylor et al., 2002No9 peanut allergic, 8 egg allergic patientsPeanutGroundNot reportedNot reportedNot reported66Not reportedNo
EggWhiteNot reportedNot reportedNot reported26.5Not reportedNo
"National Jewish" in Taylor et al., 2002No3 peanut allergic patientsPeanutGroundNot reportedNot reportedNot reported2Not reportedNo
Rance in Taylor et al., 2002No74 peanut allergic, 31 milk allergic, 38 egg allergic, 6 fish allergic patientsPeanutGroundNot reportedNot reportedNot reported0.25Not reportedNo
MilkWholeNot reportedNot reportedNot reported15Not reportedNo
EggWhole rawNot reportedNot reportedNot reported0.13Not reportedNo
FishMincedNot reportedNot reportedNot reported16mg of fishNot reportedNo
Zeiger in Taylor et al., 2002No56 milk allergic patientsMilkFormulaNot reportedNot reportedNot reported1.5Not reportedNo
Wensing et al., 2002aYes31 hazelnut allergic adultsHazelnutRaw nuts17 specified dosesYes1Subjective and ObjectiveYes
Wensing et al., 2002bYes26 peanut allergic adultsPeanutRoasted peanut meal0.03010 specified dosesNo0.1Subjective and ObjectiveYes
Fiocchi et al., 2003Yes18 children with allergy to both milk and soyMilkWhole43.24 specified dosesYes43.2ObjectiveYes
  SoyFormula21.8  21.8Objective 
Hansen et al., 2003Yes17 hazelnut allergic adultsHazelnutRaw and roasted nutsNot reportedNot reportedNot reported32 - roasted16 - rawOral allergy syndromeNo
Morisset et al., 2003YesUndefinedPeanutCrushed1.255 specified levelsYes1.25ObjectiveNo
 OilNot reportedNoNot reportedObjectiveNo
MilkLactose free0.36Yes0.36ObjectiveNo
EggRaw white0.2Yes0.2ObjectiveNo
SoyOilNot reportedNoNot reportedObjectiveNo
Osterballe and Bindslev-Jensen, 2003Yes56 egg allergic childrenEggPasteurized whole egg2.98 specified dosesYes2.9ObjectiveNo
Perry et al., 2004YesNot reportedMilk, egg, peanut, soy, wheatNot reportedNot reportedNot reportedNot reportedNot reportedNot reportedNo
Scibilia et al., 2006Yes27 wheat allergic adultsWheatFlour, raw and cooked157 specified dosesYes15ObjectiveYes

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 TypeReference aProtein Concentration (ug/g)Description of OilPublishedProtein Separation MethodProtein Quantitation Method
SoyTattrie and Yaguchi, 19730.96Refined, deodorized

Yes

ChromatographyAmino Acid Analysis
Klurfeld and Kritchevsky, 1987

1.93

0.72

Crude

"Processed"

Yes

Aqueous ExtractionCommercial Bradford Assay
Awazuhara et al., 1998

0.014

0.017

0.018

0.023

0.027

0.040

Uncharacterized, commercial

Yes

Aqueous ExtractionLowery Assay
Reeves, 1999

0.033

0.042

0.049

0.057

0.082

0.114

0.222

Fully refined, commercial

No

UnknownAmino Acid Analysis
Paschke et al., 2001

0.0332

0.0353

0.0898

0.1010

0.1380

Refined

Unrefined

Yes

Acetone PrecipitationBradford Assay
Errahali et al., 2002

0.32

1.80

Deodorized

Cold pressed

Yes

Aqueous ExtractionUnknown
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 ExtractionAmino Acid Analysis
PeanutKlurfeld and Kritchevsky, 1987

0.120

0.154

0.204

0.206

0.580

Processed

Yes

Aqueous ExtractionBradford Assay
Hoffman and Collins-Williams, 1994

0.2

0.6

3.3

3.3

Cold pressed

Yes

Aqueous ExtractionCommercial 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 ExtractionCommercial Bradford Assay
Olszewski et al., 1998

0.10

0.13

0.15

0.16

0.20

Refined, commercial

Yes

Aqueous ExtractionCommercial Bicinchoninic Acid (BCA) Assay
Skinner and Haynes, 1998187Crude

No

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

0.047

0.049

0.063

Fully refined, commercial

No

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

48

91

Refined, neutralized, bleached, deodorized

No

Aqueous ExtractionCommercial BCA Assay
220Crude   
Peeters et al., 2004

0.09

6.4

Crude, noncommercial

Yes

UnknownELISA (not described)
2.55Cold pressed   
Tree NutTeuber et al., 1997 (Almond)

2.2 ± 0.7

16.7 ± 0.8

Refined, bleached, deodorized

Yes

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

7.0 ± 2.5

7.0 ± 0.8

9.2 ± 3.1

Refined, bleached, deodorized

Yes

Aqueous ExtractionCommercial Bradford Assay
16.5 ± 2.4Unrefined   
20.4 ± 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.

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 aValidationSensitivity (LOD) (ppm gluten)Quantitation (LOQ) (ppm gluten)Raw and Baked Foods?Species SpecificityProtein(s) DetectedPracticality
Diffchamb Transia Plate GlutenNo10Not reportedNot reportedWheat, triticale, rye, barleyGliadinYes
Diffchamb Transia Plate ProlaminsWorking Group on Prolamin Analysis and Toxicity3Not reportedYesWheat, triticale, rye, barleyGliadinYes
Ingensa Gluten EIANo3Not reportedNot reportedWheat, rye, barleyGliadinNot reported
Neogen Alert for GliadinNo10NoNot reportedWheat, rye, barleyGliadinYes
Neogen Veratox for GliadinNo55Not reportedWheat, rye, barleyGliadinYes
R-BioPharm RIDASCREEN GliadinProlamin Working Group Ring Study b35YesWheat, rye, barleyGliadinYes
R-BioPharm RIDASCREEN FAST GliadinNo1010YesWheat, rye, barleyGliadinYes
R-BioPharm RIDAQUICK GliadinNo5NoYesWheat, rye, barleyGliadinYes
Tepnel BioSystems Wheat GlutenAOAC

160

2 - not validated

16YesWheat, triticale, ryeOmega gliadinYes
Tepnel BioSystems Gluten Rapid Test KitNo

50 - breads, etc

200 - "highly processed flour

NoYesWheat, triticale, ryeOmega gliadinYes

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.
StudyPublishedTest PopulationTest MaterialDose Level(s)DurationDiagnostic Assessment (Biomarker)Individual Response Data?

Fasano, 2005;

Abstract: Catassi et al., 2005b

No; analysis is ongoing33 of 46 adults completed studyNot reported0, 10 or 50 mg gluten/day3 monthsIntestinal biopsy, symptomsNot reported
Catassi et al., 1993Yes20 children (10 each dose level)Commercial crude gliadin100 mg or 500 mg gliadin/day4 weeksIntestinal biopsy, symptomsYes
Ciclitera et al., 1984Yes1 adultWhite flour milled from Kolibri strain of wheat10, 600, and 1000 mg gliadinIntraduodenal infusion over 2 hr period; 3 doses on separate daysIntestinal biopsyYes
Ciclitera et al., 1984Yes3 adultsWhite flour milled from Kolibri strain of wheat1000 mg of 4 gliadin subfractionsIntraduodenal infusion each subfraction at variable intervals of 3 to 11 daysIntestinal biopsy, symptomsYes
Ciclitera et al., 1985Yes10 adultsNot reported; article states that gluten-free bread usually contains 0.2 to 0.4 mg gliadin/30-g slice6 slices/day Juvela gluten-free bread; oral6 weeksIntestinal biopsy,symptomsYes
Montgomery et al, 1988Yes

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 AbYes, graphs
Sturgess et al, 1994Yes4 adultsUndigested gliadin prepared from Kolibri wheat flour by standard method; oligopeptides synthesized and analyzed1 gm gliadin or 200 mg of synthetic peptides/dose2 hours by infusionIntestinal biopsy hourly for 6 hrs after start infusionYes; 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