OMB First to Final Action Review (January 11, 2018)

H ALBMAYR -J ECH ET AL .: J OURNAL OF AOAC I NTERNATIONAL V OL . 98, N O . 1, 2015 105

bread) was produced as described, except that gluten-containing VWRFN ULFH ÀRXU PJ JOXWHQ NJ see above) was used. Gluten-containing crisp bread samples for the study were prepared as follows: 10 mg/kg: 17.5 g stock crisp bread was mixed with 332.5 g gluten-free crisp bread; 20 mg/kg, 35 g stock crisp bread was mixed with 315 g gluten-free crisp bread; and 100 mg/kg, 175 g stock crisp bread was mixed with 175 g gluten- free crisp bread. Mixtures were shaken in an overhead shaker for at least 1 h. The analyses of homogeneity ( see below) revealed that the gluten-free crisp bread was contaminated with gluten at a very low concentration of about 4.5 mg gluten/kg. This may have happened during production of the crisp breads, in particular during the grinding and sifting steps. Therefore, the target gluten concentrations of the crisp bread samples (0, 10, 20, and 100 mg/kg) were corrected to the gluten concentrations that were in fact present (4.5, 15, 24, and 102 mg/kg). All samples were checked for homogeneity before they were packaged in airtight bottles and accepted for the collaborative study. This was done by taking 10 representative 1 g aliquots from each bulk sample and then analyzing by the G12 Sandwich ELISA. Ideally, the CV of the 10 determinations should be 15% or less. Most samples with gluten concentration above 4 mg/kg complied with this, except the chocolate cake samples containing D ORZ FRQFHQWUDWLRQ ” PJ NJ RI LQFXUUHG JOXWHQ VKRZHG D CV of 21%. This was considered allowable for a sample like chocolate cake containing below 20 mg/kg gluten, because in an earlier study a beer (>20 mg/kg) and a starch syrup (<20 mg /kg) sample were accepted with a CV of 18–22% (7, 8). Homogeneity of Samples

cake was prepared by mixing one bag (425 g) of gluten-free cake mix (Betty Crocker Gluten-Free Cake Mix, General Mills) with 237 mL water, 112 g baking fat (Sanella, Unilever, Hamburg, Germany), and three eggs with a hand mixer at high speed for 5 min. The mass was poured into a round baking tin [diameter (Ø) = 25 cm] and baked in an oven at 170°C for 45 min. The cake was subjected to cooling for 1 h, sliced with a knife, and air-dried at room temperature (22°C) overnight (16 h). The air- dried cake was then lyophilized and ground with a household grinder (Model 836.820 1, Privileg, Fürth, Germany). Chocolate cake with a gluten concentration of 200 mg/kg (stock chocolate cake) was produced as described, except that cake mix containing ZKHDW ÀRXU FY *HQLXV ZDV XVHG 7KH DPRXQW RI ZKHDW ÀRXU LQ WKH FDNH PL[ PJ JOXWHQ NJ ZDV DGMXVWHG WR SURYLGH D ¿QDO gluten concentration of 200 mg/kg in the chocolate cake. Gluten-containing chocolate cake samples for the study were prepared as follows: 10 mg/kg, 25 g stock chocolate cake was mixed with 475 g gluten-free chocolate cake; 20 mg/kg, 50 g stock chocolate cake was mixed with 450 g gluten-free chocolate cake; and 100 mg/kg, 250 g stock chocolate cake was mixed with 250 g gluten-free chocolate cake. Mixtures were shaken in an overhead shaker for at least 1 h. The rice-based crisp bread represented a more heavily heat-treated sample. Gluten-free crisp bread was prepared by PL[LQJ J RI JOXWHQ IUHH ULFH ÀRXU see above) and 2.7 g NaCl with 270 mL of ice-cold water using a hand mixer at high speed (air incorporation). The mass was distributed in two round baking tins (25 cm diameter) to yield a dough layer of approximately 1 cm. The dough surface was perforated with a needle, and the dough was baked at 230°C for 30 min, then turned upside down and baked for another 30 min. After cooling overnight, the bread ZDV O\RSKLOL]HG DQG JURXQG WR D ¿QH SRZGHU XVLQJ D PRUWDU DQG pestle. Crisp bread containing 200 mg gluten/kg (stock crisp $JUD4XDQW *OXWHQ * DVVD\V FRQWDLQLQJ DOO WKH VWDQGDUGV ZHUH UXQ 0HDQ 2' YDOXHV 6' DQG &9 DUH VKRZQ EHORZ $OO &9 YDOXHV IRU LQWHUDVVD\ DQDO\VLV ZHUH OHVV WKDQ PHHWLQJ WKH PDQXIDFWXUHU¶V 4& FULWHULD Standard, mg/kg Mean (OD) SD (OD) CV, % 0 0.12 0.01 10.79 4 0.28 0.04 14.26 20 0.67 0.05 7.89 80 1.29 0.13 10.33 200 2.00 0.17 8.70 7DEOH 6LQJOH ODERUDWRU\ YDOLGDWLRQ GDWD RQ UHSHDWDELOLW\ XVLQJ GLIIHUHQW NLWV RI WKH VDPH EDWFK LQGLYLGXDO

Shipment

Two independent blinded replicates for each sample were provided to the participating laboratories. The coded sample vials contained 1 g of sample. Samples were shipped together with ELISA kits, instructions, and result sheet to participating laboratories.

Analysis and Data Reporting

Participants were requested to follow the instructions and to extract each sample using the test kit’s standard procedure and to analyze in duplicate in one analytical run. If changes had been made to the analytical protocol, they had to be reported in the “comments” box of the result sheet. The samples were analyzed

7DEOH /RW WR ORW YDULDWLRQ UHSURGXFLELOLW\ WKUHH GLIIHUHQW NLW EDWFKHV RI WKH $JUD4XDQW *OXWHQ * WHVW NLW ZHUH UXQ *8 *8 DQG *8 0HDQ 2' YDOXHV 6' DQG &9 DUH VKRZQ EHORZ $OO &9 YDOXHV IRU LQWHUEDWFK DQDO\VLV ZHUH RU OHVV PHHWLQJ WKH PDQXIDFWXUHU¶V 4& FULWHULD Standard, mg/kg GU1001-1106 GU1002-1108 GU1003-1111 Mean (OD) SD (OD) CV, % 0 0.10 0.10 0.09 0.10 0.01 6.97 4 0.26 0.21 0.21 0.23 0.03 11.55 20 0.72 0.57 0.58 0.63 0.08 13.45 80 1.39 1.13 1.09 1.21 0.16 13.65 200 2.11 1.65 1.60 1.79 0.28 15.92