Report EUR 21574 EN

CERTIFICATION REPORT

The Certification of Reference Materials of Dry-Mixed Maize Powder with different Mass Fractions

of MON 863 Maize

Certified Reference Material ERM®-BF416

(ERM®-BF416a/ERM®-BF416b/ERM®-BF416c/ ERM®-BF416d)

The mission of IRMM is to promote a common and reliable European measurement system in support of EU policies. European Commission Directorate-General Joint Research Centre Institute for Reference Materials and Measurements Contact information Hendrik Emons European Commission Directorate-General Joint Research Centre Institute for Reference Materials and Measurements Retieseweg 111 B-2440 Geel • Belgium Email: hendrik.emons@cec.eu.int Tel.: +32 (0)14 571 722 Fax: +32 (0)14 590 406 http://www.erm-crm.org Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server http://europa.eu.int EUR Report 21574 Luxembourg: Office for Official Publications of the European Communities ISBN 92-894-9195-7 © European Communities, 2005 Reproduction is authorised provided the source is acknowledged Printed in Belgium

Report EUR 21574 EN

CERTIFICATION REPORT

The Certification of Reference Materials of Dry-Mixed Maize Powder with different Mass Fractions

of MON 863 Maize

Certified Reference Material ERM®-BF416

(ERM®-BF416a/ERM®-BF416b/ERM®-BF416c/ ERM®-BF416d)

S. Trapmann, J. Charoud-Got, P. Conneely, M. Contreras, P. Corbisier,

D. Gancberg, E. Hannes, S. Gioria, A. Muñoz-Pineiro, M. Van Nyen, H. Schimmel, S. Szilagy, H. Emons

European Commission, Directorate General Joint Research Centre,

Institute for Reference Materials and Measurements (IRMM), Geel (BE)

1

SUMMARY This report describes the preparation and certification of dry-mixed maize powder CRMs with different mass fractions of genetically modified (GM) MON 863 maize powder (Certified Reference Materials ERM-BF416a, ERM-BF416b, ERM-BF416c and ERM-BF416d). Reference Material ERM-BF416 was originally certified as IRMM-416. The CRMs were processed in 2003 and certified in 2005 by the European Commission, Directorate General Joint Research Centre, the Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium. The CRMs are intended for the quality control and calibration of methods for the detection of genetically modified food. The MON 863 mass fractions of ERM-BF416 were verified with the help of DNA-based detection methods. The CRMs are available in glass bottles containing 1 g of maize powder packed under argon atmosphere. Seeds of non-modified maize (conventional seed line RX670) and MON 863 maize (line TP5504-TD) both supplied by Monsanto (St. Louis, MO, USA) were rinsed with demineralised water, drained and dried at 30 °C in order to minimise dust contamination from other crops. After a two step grinding process, the materials were prepared by turbula-mixing and dry-mixing of non-modified maize powder and MON 863 maize powder. ERM-BF416 was certified to contain the following MON 863 mass fractions:

CRM Certified value MON 863 mass fraction 1

[g / kg]

Uncertainty 2

[g / kg] ERM-BF 416a < 1.0 - ERM-BF 416b 1.0 -0.3 ; +1.0 ERM-BF 416c 9.8 -0.7 ; +1.2 ERM-BF 416d 98.5 -2.2 ; +2.5

1 The certified value is based on the mass fraction of dried non-genetically modified powder and dried genetically modified powder mixed and corrected for the water content. The certified value is traceable to the SI.

2 The certified uncertainty is the expanded uncertainty estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) with a coverage factor k = 2, corresponding to a level of confidence of about 95 %.

The minimum sample intake recommended for analysis is 100 mg.

2

3

Table of contents SUMMARY................................................................................................................1 TABLE OF CONTENTS............................................................................................3 GLOSSARY ..............................................................................................................4

1 INTRODUCTION.................................................................................................................5

2 CRM PREPARATION ........................................................................................................5

2.1 CHARACTERISATION OF THE BASE MATERIALS............................................................................................. 5 2.2 PROCESSING OF THE GROUND BASE MATERIALS........................................................................................... 8 2.3 QUANTITATIVE PREPARATION OF GM / NON-GM MIXTURES........................................................................ 9 2.4 BOTTLING .................................................................................................................................................... 9 2.5 PROCESSING CONTROL................................................................................................................................. 9

3 HOMOGENEITY ...............................................................................................................11

3.1 HOMOGENEITY STUDY FOR DRY-MIXED MAIZE POWDER ............................................................................ 11 3.2 MINIMUM SAMPLE INTAKE FOR ANALYSIS.................................................................................................. 12 3.2 MINIMUM SAMPLE INTAKE FOR ANALYSIS.................................................................................................. 12

4 STABILITY.........................................................................................................................12

4.1 SHORT-TERM STABILITY ............................................................................................................................ 12 4.2 LONG-TERM STABILITY .............................................................................................................................. 13

5 CERTIFIED MASS FRACTIONS AND UNCERTAINTY BUDGETS .......................13

6 VERIFICATION OF MON 863 MAIZE MIXTURES .....................................................14

REFERENCES AND ACKNOWLEDGEMENTS ............................................................17

REFERENCES.................................................................................................................................................... 17 ACKNOWLEDGEMENTS .................................................................................................................................... 18

ANNEX...................................................................................................................................19

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GLOSSARY

x average adh1 alcohol dehydrogenase 1 gene (endogenous maize gene) Bt-11 GM maize event Bt-11 Bt-176 GM maize event Bt-176 CRM certified reference material CTAB cetyltrimethylammonium bromide Ct-value number of PCR cycles to pass a set threshold CV coefficient of variance DNA deoxyribonucleic acid ERM® European Reference Material® FAM 6-FAM™ fluorescent dye GA21 GM maize event GA21 GM genetically modified GMO genetically modified organism IRMM Institute for Reference Materials and Measurements KFT Karl Fischer Titration LOD limit of detection LOQ limit of quantification MON 810 GM maize event MON 810 MON 863 GM maize event MON 863 n number of samples analysed NAA neutron activation analysis NK603 GM maize event NK603 PCR polymerase chain reaction PSA particle size analysis R2 regression coefficient RSD relative standard deviation rt-PCR real-time PCR s standard deviation SI International system of units U expanded uncertainty u uncertainty UV ultra violet

5

1 Introduction

Legislation in the European Union demands the labelling of food products consisting of or containing more than 0.9 % genetically modified organisms (GMOs), provided the GMO has been placed on the market in accordance with Community legislation [1]. This enforces the necessity on the one hand to develop and validate reliable quantitative detection methods and on the other hand to develop and produce reference materials to calibrate and control the correct application of detection methods. Therefore, mixtures of genetically modified (GM) and non-GM powders have been prepared and certified as certified reference materials CRMs. A set of CRMs of maize powder with different mass fractions of dried GM maize powder of the transformation event MON 863 (< 1.0, 1.0, 9.8, 98.5 g / kg maize) was processed and certified by IRMM. The four CRMs (ERM-BF416a, ERM-BF416b, ERM-BF416c and ERM-BF416d) are available from IRMM and its authorised distributors [2]. According to European Commission regulation (EC) No 65/2004 [3] the event MON 863 maize received the unique identifier MON-ØØ863-5. Reference Material ERM-BF416 was originally certified as IRMM-416. ERM-BF416 has been produced by means of dry-mixing techniques in order to minimise DNA and protein degradation during the processing.

2 CRM preparation

2.1 Characterisation of the base materials

For the preparation of the CRMs, seeds of non-modified maize (hybrid seed line RX670) and GM MON 863 maize (hybrid seed line TP5504-TD) were supplied to IRMM by Monsanto (St. Louis, MO, USA). The delivered non-GM and GM seed batches have been tested by Monsanto laboratories (St. Louis, MO, USA) for their purity by lateral flow immunoassay. Hundred individual GM seeds randomly chosen from the GM seed batch tested positive for the presence of the Cry3Bb protein. Additionally, four pooled samples (75 seeds each) were tested for NK603, GA21, MON 810 and MON 863 by event-specific rt-PCR. All four pools tested negative for NK603, GA21 and MON 810 and positive for MON 863. For the non-GMO seed material, four pooled samples (75 seeds each) were tested for GA21, NK603, MON 810 and MON 863 by event-specific real-time PCR (rt-PCR). The pools were negative for the events listed. It can be concluded with 95 % confidence that the non-GM batch is 99 % free of GA21, NK603, MON 810 and MON 863. Approximately 50 kg of non-modified maize and 10 kg of MON 863 maize were used for the processing of ERM-BF416. The purity and genetic composition of each batch was assessed at IRMM by real-time polymerase chain reaction (PCR) on genomic DNA extracted from leaves of individual seedlings. Seeds of each batch (n = 52) were randomly chosen and

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allowed to germinate. Genomic DNA was extracted from 110 mg pieces of the young leaves using the DNeasy® Plant Mini kit (Qiagen, Hilden, DE), analysed on a 1 % agarose gel and quantified using the PicoGreen® dsDNA quantification kit (Molecular Probes Europe, Leiden, NL). The average DNA yield ± s per 110 mg wet tissue was 29.9 ± 13.0 µg for the GM plants and 39.4 ± 14.3 µg for the non-GM plants. Detection by rt-PCR was performed at IRMM following the TaqMan® Universal PCR Master Mix protocol (Applied Biosystems, Foster City, CA, USA). Primer pairs specific for the event MON 863 and the adh1 endogenous maize gene have been used together with TaqMan probes labelled with FAM. The threshold cycle values (Ct-value) determined for the 52 GM plants were compared to a calibration curve obtained with pure MON 863 powder. All GMO plants tested positive for MON 863 and the measured average mass fraction ± s of MON 863 maize was 798 ± 82 g / kg (n = 51)1. Among the 52 plants tested one plant gave a three- to four-time increased signal. It was concluded that 51 out of the 52 plants were heterozygous for the GM event and that 1 out of 52 plants was found to be a female homozygous plant (Table 1). All non-GMO plants were tested in the same way and appeared negative for the event MON 863. Table 1: Purity test and genetic composition of the GM and non-GM seed batches used for the production of ERM-BF416

Sub- batch

PCR method performed

and primers used1

Number of

plants tested

Number of positives

Number of negatives

Non-GM event-specific real-time PCR 52 0 52 GM event-specific real-time PCR 52 522 0

1 Primer sequences of the event-specific MON 863 method have been provided by Monsanto [4] and can be found in the Annex.

2 One plant out of the 52 plants gave a three- to four-time increased GM signal when applying event-specific rt-PCR.

Additionally the purity of the ground non-GM base material was tested at IRMM. The analysis of randomly selected seeds and subsequent analysis of the powder (5 DNA extractions from 100 mg powder each) indicated that no GM contamination was detected in the non-GM lot, i.e. the values obtained were all below the detection limit (LOD) of the rt-PCR method applied (Table 2). Within the frame of an in-house validation of the method the LOD was calculated as (3.3 · s) / b, with s representing the standard deviation of a defined GM percentage and b the slope of the calibration curve. The defined GM mass fraction was the lowest GM mass fraction for which the amplification efficiency was optimal. The efficiency of the amplification was determined based on the slope of the regression line between the GM mass fraction and the Ct-values, which should not be lower than the theoretical value of 3.322. The limit of quantification (LOQ) was calculated as (10 · s) / b. Established on MON 863 powder the LOD was 0.8 g / kg and the LOQ 2.6 g / kg (Table 11). 1 Due to the calibration with powders produced from seeds (and the genetic composition of the various seed tissues), the rt-PCR results obtained for the genomic DNA extracted from plants can deviate considerably from pure GM powder.

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Table 2: Quantification of GM contamination in the non-GM base material by event-specific rt-PCR

Non-GM base material Number of DNA extractions from

100 mg powder 2

Mass fraction GM contamination 3

n [g / kg]

Event-specific rt-PCR1 5 < 0.8

1 Primer sequences of the event-specific MON 863 method have been provided by Monsanto [4] and can be found in the Annex.

2 Each rt-PCR analysis was carried out in triplicate. 3 The measured mass fraction is below the calculated LOD (see Table 11).

In order to verify that the DNA mass fraction in the GM and in the non-GM base material is the same, the DNA was extracted from the twice ground powders (as described in chapter 2.2) using a CTAB method [8] and using the Wizard® magnetic DNA purification system for food (Promega, Leiden, NL). The DNA was afterwards quantified with PicoGreen (Molecular Probes Europe, Leiden, NL) in a spectrofluorometer (Fluostar Galaxy from BMG Labtechnologies GmbH, Offenburg, DE) and by UV absorbance using a Biophotometer (Eppendorf, Hamburg, DE). The ratio between the extractable DNA mass fraction of the two materials was calculated with the following formula:

powder maize GM-non mg 100in DNA eExtractabl

powder maize 863 MON mg 100in DNA eExtractabl

A difference in DNA extractability between the two base materials was observed (Table 3). The difference proved to be significant with 95 % confidence in case of the CTAB method. In order to measure the total DNA content of both powders, a slight modification of the classical fractionation method developed initially by Ogur & Rosen [5] was employed to extract purified DNA, following the sequential removal of alcohol-, alcohol-ether- and acid-soluble compounds. After acidic digestion of the DNA fractions corresponding to the GM and non-GM powders with 1 mol / L perchloric acid, the amount of DNA was measured by a colorimetric reaction with diphenylamine, a specific reagent for 2-deoxyriboses linked to purine nucleobases [5, 6]. Using the modified method a ratio around 1 was found, indicating that the total DNA content of both materials was the same (Table 4). The user of the certified reference material should bear in mind that different extraction efficiencies of GM and non-GM powders will influence the GM mass fractions measured by rt-PCR.

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Table 3: Ratio of extractable DNA of GM and non-GM ground base material

Extraction

Method

n

Spectrofluorometer (PicoGreen)

mass ratio ± s

Biophotometer (UV radiation at 260 nm)

mass ratio ± s CTAB [8]

12 0.72 ± 0.15 0.80 ± 0.13

Wizard®

10 0.88 ± 0.32 0.87 ± 0.40

Table 4: Ratio of the total DNA content of GM and non-GM ground base material

Extraction

method

n

Spectrofluorometer (Diphenylamine) mass ratio ± s

Modified Ogur & Rosen method [5]

12

1.07 ± 0.11

2.2 Processing of the ground base materials

During the processing of ERM-BF416 the GM and non-GM ground base materials were treated separately. Cross-contamination and contamination with foreign DNA were avoided using glove box systems, clean cells, disposable laboratory clothing and treatment of all contact surfaces with a DNA destroying solution prior to exposure to the base materials. The seeds used for processing were rinsed in demineralised water, drained, and dried under vacuum at 30 °C for a minimum of 20 hours. This treatment led to a water mass fraction loss of approximately 20 g / kg. The dried seeds were then ground under Argon flushing using a high impact mill with a triangular ribbed open grinding track in order to obtain the ground base material. An additional vacuum drying at 30 °C for a minimum of 20 hours was carried out to further reduce the water content of the once ground base material with approximately 70 to 80 g / kg. For the second grinding step a sieve insert was used with 0.5 mm mesh width. Slow feeding of the mill ensured that the whole base material passed the sieve and that no selective grinding occurred. During the grinding caution was taken to avoid that the material was exposed to temperatures above 40 °C. The ground base material was mixed in a turbula mixer for 30 minutes to improve equal distribution of the different parts of the maize kernels separated by the milling process. Particle size analysis showed that both ground base materials had similar particle size distributions. Prior to dry-mixing both twice-ground base materials had a water mass fraction of approximately 22 g / kg.

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2.3 Quantitative preparation of GM / non-GM mixtures

The twice-ground base materials were used to produce powder mixtures containing nominal mass fractions of 0, 1, 10 and 100 g / kg MON 863 maize powder in non-GM maize powder. Prior to the dry-mixing, the mass fractions of the ground GM and non-GM base materials, were determined in duplicate by volumetric Karl Fischer titration (KFT, Metrohm, Berchem, BE) in order to correct for the water content of the ground base material. A 100 g / kg GM powder mixture was produced first by mixing pure GM with non-GM ground base material. All lower concentrations were achieved by further dilution of the 100 g / kg GM powder with non-GM maize powder. Ground base materials were weighed using a calibrated balance. The mass fractions were in a first step manually pre-mixed in a container and afterwards turbula mixed. The whole material was then transferred into a dry-mixing device and mixed for 2 min.

2.4 Bottling

The dry-mixed powders were bottled in cleaned 10-mL brown glass vials using an automatic sampling device. The first 30 filled bottles of each batch were discarded as an additional measure against carry-over contamination. Rubber stoppers were automatically placed on the bottle opening. Before final closure of the vials the air was evacuated in a freeze-drier and replaced with argon. The vials were closed with the help of the hydraulically moveable device of the freeze-drier and then sealed with aluminium caps to prevent opening of rubber stoppers during storage and transport. Colour-coded caps were used for easy identification of the different GM levels: nominal 0 g / kg - silver, nominal 1 g / kg - yellow, nominal 10 g / kg - red and nominal 100 g / kg - brown.

2.5 Processing control

The mass fraction of water in the powder mixtures was determined by volumetric KFT and typically amounted to values in the range of 12 to 25 g / kg (Table 5). Particle size measurements of the powders were carried out using a particle size analyser based on laser diffraction (PSA, Sympatec, Clausthal-Zellerfeld, DE). The powders had a maximum particle size below 735 µm and an average particle size around 135 µm (Table 6). Additionally, a sieving test was carried out following ISO 3310-1 using sieves with meshes of 90, 125, 180, 250, 500 and 710 µm. The average particle size determined by sieving test showed roughly similar results. It could be concluded that no particles have a particle size above 710 µm (Table 7).

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Table 5: Water mass fraction of ERM-BF416 CRM

Water mass fraction [g / kg]

n mean s ERM-BF416a 5 24.4 1.9 ERM-BF416b 5 12.2 1.4 ERM-BF416c 5 11.6 0.6 ERM-BF416d 5 17.6 1.6 Table 6: Particle size distribution of ERM-BF416, determined by laser light scattering (n = 5)

CRM Particle size distribution cumulated mass fraction

x ± s [%]

Fraction < 90 µm

Fraction < 125 µm

Fraction < 180 µm

Fraction < 255 µm

Fraction < 515 µm

Fraction < 735 µm

ERM-BF416a 40 ± 2 48 ± 3 61 ± 3 77 ± 4 99 ± 1 100 ± 0 ERM-BF416b 41 ± 2 49 ± 2 61 ± 3 77 ± 4 99 ± 2 100 ± 0 ERM-BF416c 40 ± 3 48 ± 3 62 ± 5 78 ± 6 99 ± 2 100 ± 0 ERM-BF416d 38 ± 1 46 ± 1 58 ± 2 74 ± 2 99 ± 1 100 ± 0 Table 7: Particle size distribution of ERM-BF416, determined by sieving test (n = 1)

CRM

Sample intake

Particle size distribution cumulated mass fraction

[%] [g] Fraction

< 90 µm Fraction < 125 µm

Fraction < 180 µm

Fraction < 250 µm

Fraction < 500 µm

Fraction < 710 µm

ERM-BF416a 10 23 36 50 69 100 100 ERM-BF416b 10 19 36 50 69 100 100 ERM-BF416c 10 19 35 50 69 100 100 ERM-BF416d 10 21 35 49 68 99 100 The contribution of the estimate of the particle size and the mass fraction to the uncertainty of the certified value of the CRMs has been estimated for the DNA-based methods using a software programme and DNA extractability data obtained for the various particle size fractions [9]. The influence of the particle size distribution on the uncertainty of the certified value with a given sample intake of 100 mg and an average particle size of 135 µm was estimated to be approximately 1 g / kg for a GMO CRM with a GM mass fraction of 10 g / kg.

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3 Homogeneity

3.1 Homogeneity study for dry-mixed maize powder

Prior to the processing, a homogeneity study for maize powders produced by dry-mixing was carried out by mixing Au-spiked non-GM maize powder with non-spiked non-GM maize powder. All materials used in the homogeneity study have been processed in the same way as described for the ground base materials in chapter 2.2. The 100 g / kg mass dilution was produced first and further diluted two times to reach mass fractions of 10 and 1 g / kg Au-spiked in non-spiked maize. The Au concentration of the three mixtures was determined with the help of neutron activation analysis (NAA). The results showed the homogeneity of the dry-mixed maize powder at a sample intake level of 50 mg (Table 8) and confirmed the adequacy of the dry-mixing technology for the preparation of the non-GM / GM maize mixtures. Table 8: Homogeneity study on dry-mixed Au-spiked maize powder with non-spiked maize powder, results of Au determination by neutron activation analysis (NAA) with a sample intake of 50 mg (n = 6)

Material

Mixture parts [g]

Results NAA

Au-spiked Non-spiked

Au mass fraction [µg / g]

CV [%]

Au-spiked maize 1000 0 1300 3.5 Non-spiked maize 0 1000 0.005 10.0 100 g / kg mixture 100 900 132 5.5 10 g / kg mixture 10 990 12.5 4.8 1 g / kg mixture 1 999 1.24 14.6 A homogeneity study was performed to determine the between-bottle variation, the within-bottle variation and the maximum hidden heterogeneity of CRM ERM-BF416. Five event-specific PCR measurements per bottle on five different bottles were carried out. For ERM-BF416b (nominal 1 g / kg) a relative between-bottle standard deviation of 13.49 % was found and for ERM-BF416c (nominal 10 g / kg) 9.80 %. For ERM-BF416d (nominal 100 g / kg) the relative between-bottle standard deviation was much lower than the method repeatability and it could be concluded that it was smaller than 3.2 %. The experimental data confirmed that the approach chosen for the estimation of the inhomogeneity uncertainty contribution (Table 9) was valid.

12

3.2 Minimum sample intake for analysis For the recommended sample intake of 100 mg per analysis, and taking into account the particle size distribution (average particle size 135 µm) and the mass density of the pure GM maize powder (0.89 g / mL), it was estimated that the number of particles in a 100 mg sample is larger than 8·104. Consequently 100 mg of ERM-BF416b (nominal 1 g / kg) should contain around 86 GM particles. On this basis uncertainties due to sample inhomogeneity were estimated (chapter 5). Referring to the particle size distribution it is advised to use sample intakes not smaller than 100 mg.

4 Stability

4.1 Short-term stability

In order to assess whether special care must be taken during transportation, the short-term stability of ERM-BF416 was investigated. Using an isochronous approach [10] ERM-BF416c (nominal 10 g / kg) bottles closed under Argon were exposed to +18 °C or +60 °C during 2 or 8 weeks. The DNA integrity of the samples was analysed by gel electrophoresis, the extractable DNA content was determined by UV, and the relative concentration of the transgenic sequence was verified by rt-PCR. The results were compared to results obtained for samples stored at a reference temperature of -70 °C. UV measurements and rt-PCR data confirmed that samples can be exposed for 2-3 weeks to temperatures of +60 °C (see Figure 1). Moreover, no DNA degradation was observed on the gels for any of the samples. The material will therefore be shipped under ambient conditions. Seen the stability observed at +60 °C, the data obtained for exposure at +18 °C were not evaluated.

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for rt-PCR R2 = 0.111

for extractable DNA R2 = 0.648

0102030405060708090

100110120130

0 2 4 6 8 10

weeks exposure +60 °C

mas

s o

f ext

ract

able

DN

A [n

g] /

m

ass

of p

ow

der

[mg

]

0

2

4

6

8

10

12

GM

mas

s fr

actio

n q

uan

tifie

d

by

rt-P

CR

[g /

kg]

Figure 1: Short term-stability measurements with rt-PCR (♦, normal trendline) and extractable DNA (•, dotted trendline)

4.2 Long-term stability

Earlier productions of wet-mixed maize GMO CRMs proved to have a long-term stability of at least 2.5 - 4 years provided the dried powder was stored at +4 °C in the dark and under argon. It is assumed that the stability of dry-mixed powders is higher. A post-certification monitoring is carried out at 6-months intervals in order to monitor the stability of ERM-BF416.

5 Certified mass fractions and uncertainty budgets

The materials ERM-BF416a, ERM-BF416b, ERM-BF416c and ERM-BF416d form a set of 4 reference materials certified for the mass fraction of MON 863 maize powder. The certified mass fractions are based on quantitative dry-mixing of non-modified maize powder with MON 863 maize powder. The certified value is traceable to the SI. The traceability chain to the kilogram is based on the use of calibrated balances, a thorough control of the weighing procedure and the purity of the used seeds. Taking into account the uncertainties of the weighing, the water contents of the base materials, and their purities, uncertainties for the certified mass fractions at 100 mg level were estimated (Table 9). It must, however, be emphasised that the DNA / dry powder mass fraction of different lots of maize kernels cannot be determined with high precision due to the relatively large uncertainty inherent to quantification of the total DNA content. Therefore, the ratio GM-DNA / non-GM-DNA in the reference materials may

14

significantly deviate from the certified powder mass ratio values. Well controlled production techniques in combination with sound purity controls of the GM and non-GM seeds and the derived base materials allow certifying the GM mass fractions, if the CRMs with relatively low uncertainties. Furthermore, the user of the certified reference material should bear in mind that different extraction efficiencies of GM and non-GM powders, if occurring, influence the GM concentration measured by rt-PCR. The certified value is based on the weighing of dried non-genetically modified powder and dried genetically modified powder mixed and corrected for the water content. The combined uncertainty of the certified value comprises the uncertainties introduced due to the weighing procedure, the humidity determination, the inhomogeneity at 100 mg level, and the purity of non-GM and GM base material. The uncertainty contribution of the stability testing has not been considered due to the limited method repeatability of rt-PCR and the lack of alternative methods. Table 9: Uncertainty budget for the mass fraction of MON 863 maize in ERM-BF 416

CRM,

certified mass fraction

Standard

uncertainty

Expanded uncertainty without u4

Expanded uncertainty

with u4

[g / kg] (u1)1 (u2)

2 (u3)3 (u4)

4 (u5)5 U (without u4)

6 U (with u4)7

ERM-BF416a

< 1.0 - - - - - - -

ERM-BF416b

1.0 0.0025 0.0011 0.1057 0.4850 0.0040 0.3 1.0

ERM-BF416c

9.8 0.0201 0.0087 0.3345 0.4850 0.0402 0.7 1.2

ERM-BF416d

98.5 0.1420 0.0619 1.0585 0.4850 0.4021 2.2 2.5 1 Uncertainty introduced by the mass determination (mainly based on the uncertainty of the balance). 2 Uncertainty introduced by the dilution technique. For ERM-BF416b three dilution steps, for ERM-BF416c two dilution steps and for ERM-BF416d one dilution step were taken into consideration (average of the standard deviation of the water content was 0.6 g /kg) 3 Uncertainty introduced by the inhomogeneity at 100 mg level (average particle size of 135 µm, mass density of 0.89 g / mL). 4 Uncertainty introduced by the purity of non-GM base material. 5 Uncertainty introduced by the purity of GM base material. 6 Expanded Uncertainty including the square sums of u1, u2, u3, and u5. 7 Expanded Uncertainty including the square sums of u1, u2, u3, u4, and u5.

6 Verification of MON 863 maize mixtures

The GM content of all four CRMs was verified using an event-specific rt-PCR method. The amplified PCR products are measured cycle-by-cycle with target specific reporter dyes, which lead to an increased fluorescence. The number of cycles (Ct-value) which are required to pass a fluorescence threshold correlates with the amount of target DNA in the sample. Results obtained can be found in

15

Table 10. The detection and quantification limits of the event-specific MON 863 quantification method have been established by dilution of DNA extracted from pure GM powder in nuclease free water (table 11). Table 10: Quantification by event-specific MON 863 real-time PCR [4]. DNA extracted using the CTAB method in fivefold with 100 mg powder per extraction

CRM

Certified GM mass

fraction

Expanded uncertainty

Event-specific MON 863 rt-PCR3

[g / kg] [g / kg] n2 mass fraction

[g / kg]

s

ERM-BF416a < 1.0 - 5 < 0.81 -

ERM-BF416b 1.0 -0.3 ; +1.0 5 1.0 0.2

ERM-BF416c 9.8 -0.7 ; +1.2 5 10.4 1.2

ERM-BF416d 98.5 -2.2 ; +2.5 5 111.2 3.5 1 The measured value was below the LOD of the method (see Table 11). 2 For each concentration level 5 independent extracts were analysed in five replicates. 3 Real-time PCR measures copy numbers of the targeted DNA sequence and was calibrated with known mass fractions of pure GM powder.

Table 11: Limit of detection (LOD) and limit of quantification (LOQ) of the real-time PCR methods used for the verification, established by dilution of DNA extracted from pure GM powder in non-GM DNA extracted from verified non-GM plants

Real-time PCR method

LOD

mass fraction [g / kg]

LOQ

mass fraction [g / kg]

MON 863 event-specific 0.8 2.6

Results higher than the LOD obtained with the event-specific PCR screening method (Table 10) are compared to the certified values in Figure 2. Quantification of the GM mass fraction of three mixtures of MON 863 powders by real-time PCR proved the consistency of CRM ERM-BF416. However, one has to be careful to draw quantitative conclusions from measurements of unknown samples as DNA and/or protein based GM quantification may vary with the particular maize variety tested.

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R2 = 1.00

0.1

1

10

100

1000

0.1 1 10 100 1000

GM mass fraction determined by rt-PCR [g / kg]

Cer

tifie

d G

M m

ass

frac

tion

[g /

kg]

Figure 2: Quantification of the GM mass fraction of MON 863 by event-specific real-time PCR

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References and acknowledgements

References

[1] Regulation (EC) No 1830/2003 of 22.09.2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products from genetically modified organisms and amending Directive 2001/18/EC. [2] Catalogue and sales conditions for certified reference materials: http://www.irmm.jrc.be/mrm.html. [3] Regulation (EC) No 65/2004 of 14.01.2004 establishing a system for the development and assignment of unique identifiers for genetically modified organisms. [4] Homepage of the Community Reference Laboratory for GMO Food and Feed: http://gmo-crl.jrc.it/detectionmethods.htm. [5] Ogur M., Rosen G. (1950): The Nucleic Acids of Plant Tissues. I. The Extraction and Estimation of Desoxypentose Nucleic Acid and Pentose Nucleic Acid, Plant Nucleic Acids, Archives of Biochemistry 27, 260-276. [6] Ganguli P.K. (1970): A sensitive procedure for the estimation of deoxyribonucleic acid by the diphenylamine reaction in the presence of cupric sulphate, Rev. Can. Biol. 29, 339-346. [7] Gendimenico G.J., Bouquin P.L., and Tramposch K.M. (1988): Diphenylamine-colorimetric method for DNA assay: a shortened procedure by incubating samples at 50 degrees C, Anal. Biochem. 173, 45-478. [8] Pietsch K., Waiblinger H.U., Brodmann P. and Wurz A. (1997): Screeningverfahren zur Identifizierung 'gentechnisch veränderter' pflanzlicher Lebensmittel, Deutsche Lebensmittel-Rundschau 2, 35-38. [9] Prokisch J., Zeleny R., Trapmann S., Le Guern L., Schimmel H., Kramer G.N. and Pauwels J. (2001): Estimation of the minimum uncertainty for a GM containing maize sample candidate certified reference material, Fresenius J. Anal. Chem. 370, 935-939. [10] Lamberty A., Schimmel H., and Pauwels J. (1998): The study of the stability of reference materials by isochronous measurements, Fresenius J. Anal. Chem. 360:359-361.

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Acknowledgements

The authors would like to thank Pierre Van Iwaarden, Brigitte Toussaint, and Wim Broothaerts (IRMM) for the reviewing of the certification report, as well as the experts of the Certification Advisory Panel ‘Biological Macromolecules and Biological/Biochemical Parameters’, R. Dybkaer (Frederiksberg Hospital, DK), E. Jansen (National Institutes for Public Health and Environment, NL) and U. Örnemark (EQUALIS AB, SE) for their critical comments.

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Annex

Primer and probe sequences used for the quantification of MON 863 maize [4] Sequence (5’ to 3’) MON 863 primer 1 gTA ggA TCg gAA AgC TTg gTA C MON 863 primer 2 TgT TAC ggC CTA AAT gCT gAA CT MON 863 probe 6-FAM- TgA ACA CCC ATC CgA ACA AgT Agg gTC A-TAMRA adh1 primer 1 CCA gCC TCA Tgg CCA AAg adh1 primer 2 CCT TCT Tgg Cgg CTT ATC Tg adh1 probe 6-FAM-CTT Agg ggC AgA CTC CCg TgT TCC CT-TAMRA

European Commission EUR 21574 EN – DG Joint Research Centre, Institute for Reference Materials and Measurements – The Certification of Reference Materials of Dry-Mixed Maize Powder with different Mass Fractions of MON 863 Maize - ERM®-BF416 (ERM®-BF416a/ERM®-BF416b/ERM®-BF416c/ ERM®-BF416d) Authors: S. Trapmann, J. Charoud-Got, P. Conneely, M. Contreras, P. Corbisier, D. Gancberg, E. Hannes, S. Gioria, A. Muñoz-Pineiro, M. Van Nyen, H. Schimmel, S. Szilagy, H. Emons Luxembourg: Office for Official Publications of the European Communities 2005 – 19 pp. – 21 x 29.7 cm Scientific and Technical Research series ISBN 92-894-9195-7

Abstract This report describes the preparation and certification of dry-mixed maize powder CRMs with different mass fractions of genetically modified (GM) MON 863 maize powder (European Reference Materials ERM-BF416a, ERM-BF416b, ERM-BF416c and ERM-BF416d). European Reference Material ERM-BF416 was originally certified as IRMM-416. The CRMs were processed in 2003 and certified in 2005 by the European Commission, Directorate General Joint Research Centre, the Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium. The CRMs are intended for the quality control and calibration of methods for the detection of genetically modified food. The MON 863 mass fractions of ERM-BF416 were verified with the help of DNA-based detection methods. The CRMs are available in glass bottles containing 1 g of maize powder packed under argon atmosphere. Seeds of non-modified maize (conventional seed line RX670) and MON 863 maize (line TP5504-TD) both supplied by Monsanto (St. Louis, MO, USA) were rinsed with demineralised water, drained and dried at 30 °C in order to minimise dust contamination from other crops. After a two step grinding process, the materials were prepared by turbula-mixing and dry-mixing of non-modified maize powder and MON 863 maize powder. ERM-BF416 was certified to contain the following MON 863 mass fractions:

CRM Certified value MON 863 mass fraction 1

[g / kg]

Uncertainty 2

[g / kg] ERM-BF 416a < 1.0 - ERM-BF 416b 1.0 -0.3 ; +1.0 ERM-BF 416c 9.8 -0.7 ; +1.2 ERM-BF 416d 98.5 -2.2 ; +2.5

1 The certified value is based on the mass fraction of dried non-genetically modified powder and dried genetically modified powder mixed and corrected for the water content. The certified value is traceable to the SI. 2 The certified uncertainty is the expanded uncertainty estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) with a coverage factor k = 2, corresponding to a level of confidence of about 95 %. The minimum sample intake recommended for analysis is 100 mg.

The mission of the Joint Research Centre is to provide customer-driven scientific and technical support for the conception, development, implementation and monitoring of European Union policies. As a service of the European Commission, the JRC functions as a reference centre of science and technology for the Community. Close to the policy-making process, it serves the common interest of the Member States, while being independent of special interests, whether private or national.

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LA-N

A-21574-E

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