Commission Regulation (EC) No 401/2006 of  23 February 2006  laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs   (Text with EEA relevance)

ANNEX II
ANNEX II
CRITERIA FOR SAMPLE PREPARATION AND FOR METHODS OF ANALYSIS USED FOR THE OFFICIAL CONTROL OF THE LEVELS OF MYCOTOXINS IN FOODSTUFFS
1.   INTRODUCTION
1.1.   Precautions
As the distribution of mycotoxins is generally non-homogeneous, samples shall be prepared, and especially homogenised, with extreme care.
The complete sample as received by the laboratory shall be homogenized, in case the homogenisation is performed by the laboratory.
For the analysis of aflatoxins, daylight should be excluded as much as possible during the procedure, since aflatoxin gradually breaks down under the influence of ultra-violet light.
1.2.   Calculation of proportion of shell/kernel of whole nuts
The limits fixed for aflatoxins in Regulation (EC) No 466/2001 apply to the edible part. The level of aflatoxins in the edible part can be determined by:
—
samples of nuts ‘in shell’ can be shelled and the level of aflatoxins is determined in the edible part.
—
the nuts ‘in shell’ can be taken through the sample preparation procedure. The method of sampling and analysis shall estimate the weight of nut kernel in the aggregate sample. The weight of nut kernel in the aggregate sample shall be estimated after establishing a suitable factor for the proportion of nut shell to nut kernel in whole nuts. This proportion is used to ascertain the amount of kernel in the bulk sample taken through the sample preparation and method of analysis.
Approximately 100 whole nuts shall be taken at random separately from the lot or shall be put aside from each aggregate sample. The ratio may, for each laboratory sample, be obtained by weighing the whole nuts, shelling and re-weighing the shell and kernel portions.
However, the proportion of shell to kernel may be established by the laboratory from a number of samples and so can be assumed for future analytical work. But if a particular laboratory sample is found to be in contravention of any limit, the proportion shall be determined for that sample using the approximately 100 nuts that have been set aside.
2.   TREATMENT OF THE SAMPLE AS RECEIVED IN THE LABORATORY
Each laboratory sample shall be finely grinded and mixed thoroughly using a process that has been demonstrated to achieve complete homogenisation.
In case the maximum level applies to the dry matter, the dry matter content of the product shall be determined on a part of the homogenised sample, using a method that has been demonstrated to determine accurately the dry matter content.
3.   REPLICATE SAMPLES
The replicate samples for enforcement, trade (defence) and reference (referee) purposes shall be taken from the homogenised material unless such procedure conflicts with Member States’ rules as regards the rights of the food business operator.
4.   METHOD OF ANALYSIS TO BE USED BY THE LABORATORY AND LABORATORY CONTROL REQUIREMENTS
4.1.   Definitions
A number of the most commonly used definitions that the laboratory shall be required to use are the following:
r
=
Repeatability, the value below which the absolute difference between two single test results obtained under repeatability conditions, namely same sample, same operator, same apparatus, same laboratory, and short interval of time may be expected to lie within a specific probability (typically 95 %) and hence r = 2,8 × s r .
s r
=
Standard deviation, calculated from results generated under repeatability conditions.
RSD r
=
Relative standard deviation, calculated from results generated under repeatability conditions [(s r /
) × 100].
R
=
Reproducibility, the value below which the absolute difference between single test results obtained under reproducibility conditions, namely on identical material obtained by operators in different laboratories, using the standardised test method may be expected to lie within a certain probability (typically 95 %); R = 2,8 × s R .
s R
=
Standard deviation, calculated from results under reproducibility conditions.
RSD R
=
Relative standard deviation calculated from results generated under reproducibility conditions [(s R /
) × 100].
4.2.   General requirements
Methods of analysis used for food control purposes shall comply with the provisions of items 1 and 2 of Annex III to Regulation (EC) No 882/2004.
4.3.   Specific requirements
4.3.1.   Performance criteria
Where no specific methods for the determination of mycotoxin levels in foodstuffs are required by Community legislation, laboratories may select any method provided the selected method meets the following criteria:
(a)
Performance criteria for aflatoxins
Criterion
Concentration Range
Recommended Value
Maximum permitted Value
Blanks
All
Negligible
—
Recovery — Aflatoxin M1
0,01-0,05 μg/kg
60 to 120 %
> 0,05 μg/kg
70 to 110 %
Recovery — Aflatoxins B 1 , B 2 , G 1 , G 2
< 1,0 μg/kg
50 to 120 %
1-10 μg/kg
70 to 110 %
> 10 μg/kg
80 to 110 %
Precision RSD R
All
As derived from Horwitz Equation
2 × value derived from Horwitz Equation
Precision RSD r may be calculated as 0,66 times Precision RSD R at the concentration of interest.
Note:
—
Values to apply to both B 1 and sum of B 1 + B 2 + G 1 + G 2 .
—
If sum of individual aflatoxins B 1 + B 2 + G 1 + G 2 are to be reported, then response of each to the analytical system must be either known or equivalent.
(b)
Performance criteria for ochratoxin A
Level μg/kg
Ochratoxin A
RSD r %
RSD R %
Recovery %
< 1
≤ 40
≤ 60
50 to 120
1-10
≤ 20
≤ 30
70 to 110
(c)
Performance criteria for patulin
Level μg/kg
Patulin
RSD r %
RSD R %
Recovery %
< 20
≤ 30
≤ 40
50 to 120
20-50
≤ 20
≤ 30
70 to 105
> 50
≤ 15
≤ 25
75 to 105
(d)
Performance criteria for deoxynivalenol
Level μg/kg
Deoxynivalenol
RSD r %
RSD R %
Recovery %
> 100-≤ 500
≤ 20
≤ 40
60 to 110
> 500
≤ 20
≤ 40
70 to 120
(e)
Performance criteria for zearalenone
Level μg/kg
Zearalenone
RSD r %
RSD R %
Recovery %
≤ 50
≤ 40
≤ 50
60 to 120
> 50
≤ 25
≤ 40
70 to 120
(f)
Performance criteria for Fumonisin B 1 and B 2
Level μg/kg
Fumonisin B 1 or B 2
RSD r %
RSD R %
Recovery %
≤ 500
≤ 30
≤ 60
60 to 120
> 500
≤ 20
≤ 30
70 to 110
(g)
Performance criteria for T-2 and HT-2 toxin
Level μg/kg
T-2 toxin
RSD r %
RSD R %
Recovery %
50-250
≤ 40
≤ 60
60 to 130
> 250
≤ 30
≤ 50
60 to 130
Level μg/kg
HT-2 toxin
RSD r %
RSD R %
Recovery %
100-200
≤ 40
≤ 60
60 to 130
> 200
≤ 30
≤ 50
60 to 130
(h)
Notes to the performance criteria for the mycotoxins
—
The detection limits of the methods used are not stated as the precision values are given at the concentrations of interest
—
The precision values are calculated from the Horwitz equation, i.e.:
RSD R = 2 (1-0,5logC)
where:
—
RSD R is the relative standard deviation calculated from results generated under reproducibility conditions [(s R /
) × 100]
—
C is the concentration ratio (i.e. 1 = 100g/100g, 0,001 = 1 000 mg/kg)
This is a generalised precision equation which has been found to be independent of analyte and matrix but solely dependent on concentration for most routine methods of analysis.
4.3.2.   ‘Fitness-for-purpose’ approach
In the case where there are a limited number of fully validated methods of analysis, alternatively, a ‘fitness-for-purpose’ approach, defining a single parameter, a fitness function, to evaluate the acceptability of methods of analysis may be used. A fitness function is an uncertainty function that specifies maximum levels of uncertainty regarded as fit for purpose.
Given the limited number of methods of analysis, fully validated by a collaborative trial, especially for the determination of T-2 and HT-2 toxin, the uncertainty function approach, specifying the maximum acceptable uncertainty, may also be used to assess the suitability (the ‘fitness-for-purpose’) of the method of analysis to be used by the laboratory. The laboratory may use a method which produces results within the maximum standard uncertainty. The maximum standard uncertainty may be calculated using the following formula:
where:
—
Uf is the maximum standard uncertainty (μg/kg)
—
LOD is the limit of detection of the method (μg/kg)
—
α is a constant, numeric factor to be used depending on the value of C. The values to be used are set out in the table hereafter
—
C is the concentration of interest (μg/kg).
If the analytical method provides results with uncertainty measurements less than the maximum standard uncertainty the method shall be considered being equally suitable to one which meets the performance criteria given in point 4.3.1.
Table
Numeric values to be used for α as constant in formula set out in this point, depending on the concentration of interest
C (μg/kg)
α
≤ 50
0,2
51-500
0,18
501-1 000
0,15
1 001 -10 000
0,12
> 10 000
0,1
4.4.   Estimation of measurement uncertainty, recovery calculation and reporting of results  ( 1 )
The analytical result must be reported corrected or uncorrected for recovery. The manner of reporting and the level of recovery must be reported. The analytical result corrected for recovery shall be used for controlling compliance.
The analytical result must be reported as x +/– U whereby x is the analytical result and U is the expanded measurement uncertainty.
U is the expanded measurement uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %.
For food of animal origin, the taking into account of the measurement uncertainty can also be done by establishing the decision limit (CCα) in accordance with Commission Decision 2002/657/EC  ( 2 ) (point 3.1.2.5. of the Annex — the case of substances with established permitted limit).
The present interpretation rules of the analytical result in view of acceptance or rejection of the lot apply to the analytical result obtained on the sample for official control. In case of analysis for defence or referee purposes, the national rules apply.
4.5.   Laboratory quality standards
Laboratory must comply with the provisions of Article 12 of Regulation (EC) No 882/2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules  ( 3 ) .
( 1 )   More details on procedures for the estimation of measurement uncertainty and on procedures for assessing recovery can be found in the report ‘Report on the relationship between analytical results, measurement uncertainty, recovery factors and the provisions of EU food and feed legislation’ — http://europa.eu.int/comm/food/food/chemicalsafety/contaminants/report-sampling_analysis_2004_en.pdf
( 2 )
OJ L 221, 17.8.2002, p. 8 . Decision as last amended by Decision 2004/25/EC ( OJ L 6, 10.1.2004, p. 38 ).
( 3 )   See also the transitional arrangements provided for in article 18 of Commission Regulation (EC) No 2076/2005 of 5 December 2005 laying down transitional arrangements for the implementation of Regulation (EC) No 853/2004, 854/2004 and 882/2004 of the European Parliament and of the Council and amending Regulations (EC) No 853/2004 and 854/2004 ( OJ L 338, 22.12.2005, p. 83 ).
Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs (Text with EEA relevance)

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