Commission Implementing Regulation (EU) 2023/2782 of 14 December 2023 laying down the methods of sampling and analysis for the control of the levels of mycotoxins in food and repealing Regulation (EC) No 401/2006

This method of sampling is of application for the official control of the levels of mycotoxins in solid processed fruit (with the exception of processed products from dried fruit which fall under parts B and C of this Annex) and vegetable products, including solid processed fruit and vegetable products for infants and young children.

I.1.   Method of sampling

The aggregate sample shall be at least 1 kg, except where it is not possible e.g. when sampling a single package.

The minimum number of incremental samples to be taken from the lot shall be as given in Table 1.

The incremental samples shall be of similar weight. The weight of an incremental sample shall be at least 100 g, resulting in an aggregate sample of at least 1 kg. Departure from this method shall be recorded in the record provided for under part I point A.1.8 of this Annex.

Table 1

Minimum number of incremental samples to be taken from the lot

Weight of lot (in kg)	Minimum number of incremental samples to be taken	Aggregate sample weight(kg)
< 50	3	1
50 to 500	5	1
> 500	10	1

If the lot consists of individual packages, then the number of packages, which shall be taken to form the aggregate sample, is given in Table 2.

Table 2

Number of packages (incremental samples) which shall be taken to form the aggregate sample if the lot consists of individual packages

Number of packages or units in the lot	Number of packages or units to be taken	Aggregate sample weight(kg)
1 to 25	1 package or unit	1
26 to 100	about 5  %, at least two packages or units	1
> 100	about 5  %, at maximum 10 packages or units	1

I.2.   Sampling at retail stage

Sampling of foodstuffs at the retail stage shall be done where possible in accordance with the sampling provisions set out in this part I.

Where that is not possible, an alternative method of sampling at retail stage may be used provided that it ensures that the aggregate sample is sufficiently representative of the sampled lot and is fully described and documented ([^E0020]).

I.3.   Acceptance of a lot or sublot

• — acceptance if the laboratory sample conforms to the maximum level, taking into account the measurement uncertainty and correction for recovery,

• — rejection if the laboratory sample exceeds the maximum level beyond reasonable doubt taking into account the measurement uncertainty and correction for recovery. This is the case when the analytical result (corrected for recovery if applicable) minus the expanded measurement uncertainty arising from the analysis is above the maximum level.

J. METHOD OF SAMPLING FOR BABY FOODS AND PROCESSED CEREAL BASED FOODS FOR INFANTS AND YOUNG CHILDREN

This method of sampling is of application for the official control of the levels of mycotoxins in baby foods and processed cereal based foods for infants and young children, with the exception of beverages referred to in part H and solid processed fruit and vegetable products referred to in part I of this Annex.

J.1.   Method of sampling

• — The method of sampling for cereals and cereal products as set out in point A.4 in part II of this Annex shall apply to food intended for infants and young children. Accordingly, the number of incremental samples to be taken shall depend on the weight of the lot, with a minimum of 10 and a maximum of 100, in accordance with Table 2 at point A.4 in part II of this Annex. For very small lots (≤ 0,5 tonnes) a lower number of incremental samples may be taken, but the aggregate sample uniting all incremental samples shall be also in that case at least 1 kg.

• — weight of the incremental sample shall be about 100 g. In the case of lots in retail/individual packages, the weight of the incremental sample shall depend on the weight of the retail/individual package and in case of very small lots (≤ 0,5 tonnes) the incremental samples shall have a weight as such that uniting the incremental samples results in an aggregate sample of at least 1 kg. Departure from this method shall be recorded in the record provided for under part I point A.1.8 of this Annex.

• — weight of aggregate sample = 1-10 kg sufficiently mixed.

J.2.   Sampling at retail stage

Sampling of foodstuffs at the retail stage shall be done where possible in accordance with the sampling provisions set out in this part J.

Where that is not possible, an alternative method of sampling at retail stage may be used provided that it ensures that the aggregate sample is sufficiently representative of the sampled lot and is fully described and documented ([^E0021]).

J.3.   Acceptance of a lot or sublot

• — acceptance if the laboratory sample conforms to the maximum level, taking into account the correction for recovery and measurement uncertainty;

• — rejection if the laboratory sample exceeds the maximum level beyond reasonable doubt taking into account the correction for recovery and measurement uncertainty. This is the case when the analytical result (corrected for recovery if applicable) minus the expanded measurement uncertainty arising from the analysis is above the maximum level.

K. METHOD OF SAMPLING FOR VEGETABLE OILS

K.1.   Method of sampling for vegetable oils

• — The weight of the incremental sample shall be at least about 100 g (ml) (depending of the nature of the lot e.g. vegetable oil in bulk, at least 3 incremental samples of about 350 ml have to be taken), resulting in an aggregate sample of at least 1 kg (litre).

• — On condition that the sublot can be separated physically, each lot shall be subdivided into sublots following Table 1. Taking into account that the weight of the lot is not always an exact multiple of the weight of the sublots, the weight of the sublot may exceed the mentioned weight by a maximum of 20 %. In case the lot is not or cannot be physically separated into sublots, a minimum of 3 incremental samples is taken from the lot.

• — The minimum number of incremental samples to be taken from the lot shall be as given in Table 2. The lot shall be thoroughly mixed insofar possible by either manual or mechanical means immediately prior to sampling. In this case, a homogeneous distribution of mycotoxins can be assumed within a given lot, it is therefore sufficient to take three incremental samples from a lot to form the aggregate sample. Table 1 Subdivision of lots into sublots depending on lot weight Commodity Lot weight (tonne) Weight or number of sublots Minimum No incremental samples Minimum aggregate sample weight (kg) Vegetable oils ≥ 1 500 500 tonnes 3 1

  300 and < 1 500 3 sublots 3 1 ≥ 50 and ≤ 300 100 tonnes 3 1 < 50 — 3 1 Table 2 Minimum number of incremental samples to be taken from the lot Form of commercialisation Weight of lot (in kg) Volume of lot (in litres) Minimum number of incremental samples to be taken Bulk [^E0022] — 3 packages ≤ 50 3 packages > 50 to 500 5 packages > 500 10 (^*1) On condition that the sublot can be separated physically, large bulk lots of vegetable oils shall be subdivided into sublots as foreseen in Table 2 of this part K

K.2.   Method of sampling for vegetable oils at retail stage

Sampling of foodstuffs at the retail stage shall be done where possible in accordance with the sampling provisions set out in this part K.

Where that is not possible, other effective methods of sampling at retail stage may be used provided that they ensure that the aggregate sample is sufficiently representative of the sampled lot and is fully described and documented. In any case, the aggregate sample shall be at least 1 kg.

K.3.   Acceptance of a lot or sublot

• — acceptance if the laboratory sample conforms to the maximum level, taking into account the correction for recovery and measurement uncertainty,

• — rejection if the laboratory sample exceeds the maximum level beyond reasonable doubt taking into account the correction for recovery and measurement uncertainty. This is the case when the analytical result (corrected for recovery if applicable) minus the expanded measurement uncertainty arising from the analysis is above the maximum level.

L. METHOD OF SAMPLING FOR FOOD SUPPLEMENTS, POLLEN AND POLLEN PRODUCTS

L.1.   Weight of incremental sample and method of sampling

The sampling procedure provided for food supplements, pollen and pollen products as capsules/pills is based on retail/individual packages containing usually 30 to 120 capsules/pills per retail/individual package

Lot size (number of retail/individual packages)	Number of retail/individual packages to be taken for sample	Sample size (minimum amount of the aggregate sample)
1 -50	1	Food supplements as capsules/pills: Total content of the retail/individual package
1 -50	1	Other forms of food supplements – incremental samples of approx. 20 g or 20 ml — 100 g for food supplements containing herbal/plant based ingredients including extracts (minimum 5 incremental samples) — 50 g or 50 ml for other food supplements (minimum 3 incremental samples)
51 -250	2	Food supplements as capsules/pills: total content of the two retail/individual packages
51 -250	2	Other forms of food supplements – incremental samples of approx. 20 g or 20 ml — 200 g for food supplements containing herbal/plant based ingredients including extracts (minimum 10 incremental samples) — 100 g or 100 ml for other food supplements (minimum 5 incremental samples)
251 -1 000	4	Food supplements as capsules/pills: from each retail/individual package taken for sample, half of the capsules/pills
251 -1 000	4	Other forms of food supplements – incremental samples of approx. 20 g or 20 ml — 200 g for food supplements containing herbal/plant based ingredients including extracts (minimum 10 incremental samples) — 100 g or 100 ml for other food supplements (minimum 5 incremental samples)
> 1 000	4 + 1 retail/individual packages per 1 000 retail/individual packages with a maximum of 25 retail/individual packages	Food supplements as capsules/pills: ≤ 10 retail/individual packages: from each retail/individual package, half of the capsules/pills > 10 retail/individual packages: from each retail/individual package, an equal number of capsules/pills is taken to result in a sample with the equivalent of the content of retail/individual 5 packages
> 1 000	4 + 1 retail/individual packages per 1 000 retail/individual packages with a maximum of 25 retail/individual packages	Other forms of food supplements – incremental samples of approx. 20 g or 20 ml ≤ 10 retail/individual packages: — 200 g for food supplements containing herbal/plant based ingredients including extracts (minimum 10 incremental samples) — 100 g or 100 ml for other food supplements (minimum 5 incremental samples) > 10 retail/individual packages – per 5 retail/individual packages: — 100 g for food supplements containing herbal/plant based ingredients including extracts (minimum 5 incremental samples) — 50 g or 50 ml for other food supplements (minimum 3 incremental samples)
Unknown (only applicable for e-commerce)	1	Food supplements as capsules/pills: total content of the package

L.2.   Sampling at retail

Sampling of food supplements, pollen and pollen products at the retail stage shall be done where possible in accordance with the sampling provisions set out in this part L.

Where that is not possible, an alternative method of sampling at retail stage may be used provided that it ensures that the aggregate sample is sufficiently representative of the sampled lot and is fully described and documented. In any case, the aggregate sample shall be at least 0,05 kg.

L.3.   Acceptance of a lot

• — acceptance if the laboratory sample conforms to the maximum level, taking into account the correction for recovery and measurement uncertainty;

• — rejection if the laboratory sample exceeds the maximum level beyond reasonable doubt taking into account the correction for recovery and measurement uncertainty. This is the case when the analytical result (corrected for recovery if applicable) minus the expanded measurement uncertainty arising from the analysis is above the maximum level.

M. METHOD OF SAMPLING FOR DRIED HERBS, HERBAL INFUSIONS (DRIED PRODUCT), TEAS (DRIED PRODUCT) AND POWDERED SPICES

M.1.   Weight of the incremental sample

The weight of the incremental sample shall be about 80 g, unless otherwise defined in this part M.

In the case of lots in retail/individual packages, the weight of the incremental sample depends on the weight of the retail/individual package.

In the case of retail/individual package of > 80 g, this will result in aggregate samples weighing more than the required weight indicated in tables 1 and 2. If the weight of a single retail/individual package is >> 80 g, then 80 g shall be taken from each individual retail/individual package as an incremental sample. This can be done either when the sample is taken or in the laboratory. However, in cases where such method of sampling would lead to unacceptable commercial consequences resulting from damage to the lot (because of packaging forms, means of transport, etc.), an alternative method of sampling can be applied. For example, in case where a valuable product is marketed in retail/individual packages of 500 g or 1 kg, the aggregate sample can be obtained by the aggregation of a number of incremental samples that is smaller than the number indicated in Tables 1 and 2, on the condition that the weight of the aggregate sample corresponds to the required weight of the aggregate sample mentioned in Tables 1 and 2.

Where the retail/individual packages are less than 80 g and if the difference is small (i.e. not less than half of 80 g), one retail/individual package shall be considered as one incremental sample, resulting in an aggregate sample of less than the required weight indicated in tables 1 and 2. If the weight of the retail/individual packages are much less than 80 g, one incremental sample shall consist of two or more retail/individual packages, whereby the 80 g are approximated as closely as possible.

M.2.   Division of lots into sublots for the sampling of dried herbs, herbal infusions (dried product), teas (dried product) and powdered spices

Table 1

Subdivision of lots into sublots depending on lot weight

Commodity	Lot weight (tonnes)	Weight of sublots	Number of incremental samples	Aggregate sample weight (kg)
dried herbs, herbal infusions (dried product) teas (dried product), powdered spices	≥ 15	25 tonnes	50	4
dried herbs, herbal infusions (dried product) teas (dried product), powdered spices	< 15	—	3 – 50 [^E0023]	0,2 – 4,0

Depending on the lot weight – see Table 2.

M.3.   Method of sampling for dried herbs, herbal infusions (dried product), teas (dried product) and powdered spices (lots ≥ 15 tonnes)

On condition that the sublot can be separated physically, each lot shall be subdivided into sublots following Table 1. Taking into account that the weight of the lot is not always an exact multiple of the weight of the sublots, the weight of the sublot may exceed the mentioned weight by a maximum of 20 %.

Each sublot shall be sampled separately.

The number of incremental samples shall be 50. The weight of the aggregate sample shall be 4,0 kg.

If it is not possible to carry out the method of sampling described above because of the unacceptable commercial consequences resulting from damage to the lot (because of packaging forms, means of transport or other reasons) an alternative method of sampling may be applied provided that it is as representative as possible and is fully described and documented.

M.4.   Method of sampling for dried herbs, herbal infusions (dried product) and teas (dried product) and powdered spices (lots < 15 tonnes)

For lots of dried herbs, herbal infusions (dried product) and teas (dried product) and powdered spices of less than 15 tonnes the sampling plan shall consist in 3 to 50 incremental samples, depending on the lot weight, resulting in an aggregate sample of 0,2 to 4,0 kg.

The figures in the following Table 2 may be used to determine the number of incremental samples to be taken.

Table 2

Minimum number of incremental samples to be taken depending on the weight of the lot of dried herbs, herbal infusions (dried product), teas (dried product) and powdered spices

Lot weight (tonnes)	Minimum number of incremental samples	Minimum aggregate sample weight (kg)
≤ 0,1	3	0,2
> 0,1 – ≤ 0,5	10	0,8
> 0,5 – ≤ 5,0	25	2,0
> 5,0 – ≤ 10,0	35	2,8
> 10,0 – ≤ 15,0	50	4,0

M.5.   Sampling at retail stage

Sampling of foodstuffs at the retail stage shall be done where possible in accordance with the sampling provisions set out in this part M.

Where this is not possible, an alternative method of sampling at retail stage may be used provided that it ensures that the aggregate sample is sufficiently representative of the sampled lot and is fully described and documented. In any case, the aggregate sample shall weigh at least 0,2 kg.

M.6.   Sampling of food supplements containing dried herbs in bulk before their packaging in retail/individual packages for the final consumer

For the sampling of food supplements containing dried herbs in bulk before their packaging in retail/individual packages, the sampling provisions provided for in this part M apply and shall be applied based on the weight of the dried herb content of the food supplements in bulk.

M.7.   Acceptance of a lot or sublot

Acceptance: where the laboratory sample conforms to the maximum level, taking into account the correction for recovery and measurement uncertainty.

Rejection: where the laboratory sample exceeds the maximum level beyond reasonable doubt taking into account the correction for recovery and measurement uncertainty. This is the case when the analytical result (corrected for recovery if applicable) minus the expanded measurement uncertainty arising from the analysis is above the maximum level.

N. METHOD OF SAMPLING FOR VERY LARGE LOTS OR LOTS STORED OR TRANSPORTED IN A WAY WHEREBY SAMPLING THROUGHOUT THE LOT IS NOT FEASIBLE

N.1.   General principles

In case the way of transport or storage of a lot does not enable to take incremental samples throughout the whole lot, sampling of such lots shall preferably be done when the lot is in flow (dynamic sampling).

In the case of large warehouses destined to store food, operators shall be encouraged to install equipment in the warehouse enabling (automatic) sampling across the whole stored lot.

When a sampling procedure as provided for in this part N is applied, the food business operator or his representative shall be informed of the sampling procedure. If the sampling procedure is questioned by the food business operator or his representative, the food business operator or his representative shall enable the competent authority to sample throughout the whole lot at his/her own cost.

Sampling of a portion of the lot is allowed, on the condition that the quantity of the sampled portion is at least 10 % of the lot to be sampled. If a portion of a lot of food of the same class or description has been sampled and identified as not satisfying Union requirements, it shall be presumed that the entire lot is also affected, unless further detailed assessment shows no evidence that the rest of the lot is unsatisfactory.

The relevant sampling provisions, such as weight of the incremental sample, provided for in the other parts of this Annex are applicable for the sampling for very large lots or lots stored or transported in a way whereby sampling throughout the lot is not feasible.

N.2.   Number of incremental samples to be taken in the case of very large lots

In the case of large sampled portions (sampled portions > 500 tonnes), the number of incremental samples to be taken = 100 incremental samples + √tonnes. However, in case the lot is less than 1 500 tonnes and can be subdivided into sublots in accordance with the Table 1 of part A and on the condition that the sublots can be separated physically, the number of incremental samples as provided for in part A have to be taken.

N.3.   Large lots transported by ship

N.3.1.   Dynamic sampling of large lots transported by ship

The sampling of large lots in ships is preferably carried out while the product is in flow (dynamic sampling).

The sampling is to be done per hold (entity that can physically be separated). Holds are however emptied partly one after the other so that the initial physical separation no longer exists after transfer into storage facilities. Sampling can therefore be performed based on initial physical separation or based on the separation after transfer into the storage facilities.

The unloading of a ship can last for several days. Normally, sampling has to be performed at regular intervals during the whole duration of unloading. It is however not always feasible or appropriate for an official inspector to be present for sampling during the whole operation of unloading. Therefore, sampling of a portion of the lot is allowed to be undertaken (sampled portion). The number of incremental samples is determined by taking into account the size of the sampled portion.

Even if the official sample is taken automatically, the presence of an inspector is necessary. However, if the automatic sampling is done with pre-set parameters which cannot be changed during the sampling and the incremental samples are collected in a sealed receptacle, preventing any possible fraud, then the presence of an inspector is only required at the beginning of the sampling, every time the receptacle of the samples needs to be changed and at the end of the sampling.

N.3.2.   Sampling of lots transported by ship by static sampling

In cases where the sampling is done in a static way the same procedure as foreseen for storage facilities (silos) accessible from above has to be applied (see point N.5.1).

The sampling has to be performed on the accessible part (from above) of the lot/hold. The number of incremental samples is determined by taking into account the size of the sampled portion.

N.4.   Sampling of large lots stored in warehouses

The sampling has to be performed on the accessible part of the lot. The number of incremental samples is determined by taking into account the size of the sampled portion.

N.5.   Sampling of storage facilities (silos)

N.5.1.   Sampling of silos (easily) accessible from above

The sampling has to be performed on the accessible part of the lot. The number of incremental samples is determined by taking into account the size of the sampled portion.

N.5.2.   Sampling of silos not accessible from above (closed silos)

N.5.2.1.   Silos not accessible from above (closed silos) with individual sizes > 100 tonnes

Food stored in such silos cannot be sampled in a static way. Therefore, when the food in the silo has to be sampled and there is no possibility to move the lot, an agreement has to be made with the operator that he or she has to inform the inspector about when the silo will be unloaded, partially or completely, in order to enable sampling when the food is in flow.

N.5.2.2.   Silos not accessible from above (closed silos) with individual sizes < 100 tonnes

Contrary to the provision in point N.1 (sampled portion at least 10 %), the sampling procedure involves the release into a receptacle of a quantity of 50 to 100 kg and taking the sample from it. The size of the aggregate sample corresponds to the whole lot and the number of incremental samples relate to the quantity of the food from the silo released into the receptacle for sampling.

N.6.   Sampling of loose food in large closed containers

Such lots can often only be sampled when unloaded. In certain cases it is not possible to unload at the point of import or control and therefore the sampling should take place when such containers are unloaded. The operator has to inform the inspector about the place and time of unloading the containers to enable the inspector to be present.

ANNEX II

Criteria for sample preparation and for methods of analysis used for the control of the levels of mycotoxins in food

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 homogenised, in case the homogenisation is performed by the laboratory.

For the analysis of aflatoxins, daylight shall 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/oilseeds (peanuts and other)

The maximum levels established in Regulation (EU) 2023/915 apply to the edible part. The level of mycotoxins in the edible part can be determined by:

• — samples of nuts and oilseeds ‘in shell’ can be shelled and the level of mycotoxins is determined in the edible part;

• — the nuts and oilseeds ‘in shell’ can be taken through the sample preparation procedure. The method of sampling and analysis shall estimate the weight of kernel in the aggregate sample. The weight of kernel in the aggregate sample shall be estimated after establishing a suitable factor for the proportion of shell to kernel in whole nuts and oilseeds. This proportion is used to ascertain the amount of kernel in the aggregate sample taken through the sample preparation and method of analysis.

Approximately 100 whole nuts/oilseeds 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 and oilseeds, 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 maximum level, the proportion shall be determined for that sample using the approximately 100 nuts/oilseeds that have been set aside.

2. TREATMENT OF THE SAMPLE AS RECEIVED IN THE LABORATORY

Each laboratory sample shall be mixed thoroughly using a process, including fine grinding if needed, that has been demonstrated to achieve complete homogenisation with the exception of samples for the control of the presence of ergot sclerotia.

In case the laboratory sample has to be analysed for the control of the presence of ergot sclerotia and mycotoxins, the part of the sample used for the determination of ergot sclerotia is taken from the laboratory sample before grinding of the laboratory sample.

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, defence and reference purposes shall be taken from the homogenised aggregate sample 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.   General requirements

Confirmatory methods of analysis used for food control purposes shall comply with the provisions of points 1 and 2 of Annex III to Regulation (EU) 2017/625.

Wherever possible, the trueness of the method should be verified by analysis of a certified reference material and/or successful participation in proficiency tests on a regular basis.

4.2.   Specific requirements

4.2.1.   Specific requirements for confirmatory methods

4.2.1.1.   Performance criteria

For confirmatory methods the following performance criteria apply:

Recovery: the average recovery should be between 70 and 120 %.

The average recovery is the average value from replicates obtained during validation when determining the precision parameters RSDr and RSDwR. The criterion applies to all concentrations and all individual toxins, with the exception of ergot alkaloids.

For ergot alkaloids the criterion applies to the sum of each epimer-pair.

In exceptional cases, average recoveries outside the above range can be acceptable but shall lie within 50-130 %, and only when the precision criteria for RSDr and RSDwR are met.

Precision

• RSDr shall be ≤ 20 %.

• RSDwR shall be ≤ 20 %.

• RSDR should be ≤ 25 %.

These criteria apply to all concentrations.

In case a laboratory provides the evidence that the RSDwR criterion is complied with, there is no need to provide that evidence for the RSDr criterion as compliance with the RSDwR guarantees compliance with the RSDr criterion.

In case the maximum level applies to a sum of toxins, then the criteria for precision apply to both the sum and the individual toxins. For ergot alkaloids, the criteria for individual toxins apply to the sum of each epimer pair.

Limit of quantification

When a specific requirement for the LOQ of a mycotoxin has been set in the Table 1 below, the method shall have an LOQ at or below this value.

Table 1

LOQ requirements for certain mycotoxins

Mycotoxin	Food	LOQ requirement (μg/kg)
Aflatoxins
Aflatoxin B1	Baby food and processed cereal-based foods for infants and young children, and food for special medical purposes intended for infants and young children	≤ 0,1
Aflatoxin B1, B2, G1, G2, each of the aflatoxins	All other foods	≤ 1
Ochratoxin A	Liquorice confectionary containing < 97 % liquorice extract on dry basis	≤ 10,0
	Cocoa powder	≤ 3,0
Ergot alkaloids (each of 12 epimers included in sum definition of ML)	Cereals and cereal-based foods	≤ 4
	Processed cereal-based food for infants and young children	≤ 2

In all other cases, the following applies:

LOQ: hall be ≤ 0,5ML and should preferably be lower (≤ 0,2ML).

In case the maximum level applies to a sum of toxins, then the LOQ of the individual toxins shall be ≤ 0,5*ML/n, with n being the number of toxins included in the ML definition.

Identification

For identification, the criteria as laid down in the Guidance document on identification of mycotoxins and plant toxins in food and feed ([^E0024]) shall be applied.

4.2.1.2.   Extension of the scope of the method

4.2.1.2.1.   Extension of scope to other mycotoxins:

When additional analytes are added to the scope of an existing confirmatory method, a full validation is required to demonstrate the suitability of the method.

4.2.1.2.2.   Extension to other commodities:

If the confirmatory method is known or expected to be applicable to other commodities, the validity to these other commodities shall be verified. As long as the new commodity belongs to a commodity group (see Table 2 in this Annex) for which an initial validation has already been performed, a limited additional validation is sufficient.

4.2.2.   Specific requirements for semi-quantitative screening methods

4.2.2.1.   Scope

This section applies to bioanalytical methods based on immuno-recognition or receptor binding (such as ELISA, dip-sticks, lateral flow devices, immuno-sensors) and physicochemical methods based on chromatography or direct detection by mass spectrometry (e.g. ambient MS). Other methods (e.g. thin layer chromatography) are not excluded provided the signals generated relate directly to the mycotoxins of interest and allow that the principle described hereunder is applicable.

The specific requirements apply to methods of which the result of the measurement is a numerical value, for example a (relative) response from a dip-stick reader, a signal from LC-MS, etc., and that normal statistics apply.

The requirements do not apply to methods that do not give numerical values (e.g. only a line that is present or absent), which require different validation approaches. Specific requirements for these methods are provided in point 4.2.3.

This document describes procedures for the validation of screening methods by means of an inter-laboratory validation, the verification of the performance of a method validated by means of an inter-laboratory exercise and the single-laboratory validation of a screening method.

4.2.2.2.   Validation procedure

The aim of the validation is to demonstrate the fitness of purpose of the screening method. This is done by determination of the cut-off value and determination of the false negative and false suspect rate. In these two parameters performance characteristics such as detection capability, selectivity, and precision are embedded.

Screening methods may be validated by inter-laboratory or by single laboratory validation. If inter-laboratory validation data is already available for a certain mycotoxin/matrix/STC combination, a verification of method performance is sufficient in a laboratory implementing the method.

4.2.2.2.1.   Initial validation by single laboratory validation

Mycotoxins

The validation shall be performed for every individual mycotoxin in the scope. In case of bio-analytical methods that give a combined response for a certain mycotoxin group (e.g. aflatoxins B1, B2, G1 & G2; fumonisins B1 & B2), applicability shall be demonstrated and limitations of the test mentioned in the scope of the method. Undesired cross-reactivity (e.g. DON-3-glycoside, 3- or 15-acetyl-DON for immuno-based methods for DON) is not considered to increase the false negative rate of the target mycotoxins, but may increase the false suspect rate. This unwanted increasing shall be diminished by confirmatory analysis for unambiguous identification and quantification of the mycotoxins.

Matrices

An initial validation shall be performed for each commodity, or, when the method is known to be applicable to multiple commodities, for each commodity group. In the latter case, one representative and relevant commodity shall be selected from that group (see Table 2).

Sample set

The minimum number of different samples required for validation is 20 homogeneous negative control samples and 20 homogeneous positive control samples that contain the mycotoxin at the STC, analysed under intermediate precision (RSDRi) conditions spread over 5 different days. Additional sets of 20 samples containing the mycotoxin at other levels may be added to the validation set to gain insight as to what extent the method can distinguish between different mycotoxin concentrations.

Concentration

For each STC to be used in routine application, a validation shall be performed.

4.2.2.2.2.   Initial validation through collaborative trials

Validation through collaborative trials shall be done in accordance with ISO 5725:1994 or the IUPAC International Harmonised Protocol or other internationally recognised protocol on collaborative trials which requires inclusion of valid data from at least eight different laboratories. The only other difference compared to single laboratory validations shall be that the ≥ 20 samples per commodity/level may be evenly divided over the participating laboratories, with a minimum of two samples per laboratory.

4.2.2.3.   Determination of cut-off value and rate of false suspected results of blank samples

The (relative) responses for the negative control and positive control samples shall be taken as basis for the calculation of the required parameters.

Screening methods with a response proportional with the mycotoxin concentration

For screening methods with a response proportional with the mycotoxin concentration the following applies:

Cut-off value = RSTC – t-value0,05 SDSTC*

RSTC = mean response of the positive control samples (at STC)

t-value: = one tailed t-value for a rate of false negative results of 5 % (see Table 3)

SDSTC = standard deviation

Screening methods with a response inversely proportional with the mycotoxin concentration

Similarly, for screening methods with a response inversely proportional with the mycotoxin concentration, the cut-off value is determined as:

Cut-off value = RSTC + t-value0,05 SDSTC*

By using this specific t-value for determining the cut-off value, the rate of false negative results is by default set at 5 %.

Fitness for purpose assessment

Results from the negative control samples are used to estimate the corresponding rate of false suspect results. The t-value is calculated corresponding to the event that a result of a negative control sample is above the cut-off value, thus erroneously classified as suspect.

t-value = (cut-off value – meanblank)/SDblank

for screening methods with a response proportional with the mycotoxin concentration

or

t-value = (meanblank – cut-off value)/SDblank

for screening methods with a response inversely proportional with the mycotoxin concentration.

From the obtained t-value, based on the degrees of freedom calculated from the number of experiments, the probability of false suspect samples for a one tailed distribution can either be calculated (e.g. spread sheet function ‘TDIST’) or taken from a table for t-distribution (see Table 3).

The corresponding value of the one tailed t-distribution specifies the rate of false suspect results.

This concept is described in detail with an example in Analytical and Bioanalytical Chemistry DOI 10.1007/s00216 -013-6922-1.

4.2.2.4.   Extension of the scope of the method

4.2.2.4.1.   Extension of scope to other mycotoxins:

When additional analytes are added to the scope of an existing screening method, a full validation shall be required to demonstrate the suitability of the method.

4.2.2.4.2.   Extension to other commodities:

If the screening method is known or expected to be applicable to other commodities, the validity to these other commodities shall be verified. As long as the new commodity belongs to a commodity group (see Table 2 in this Annex) for which an initial validation has already been performed, a limited additional validation is sufficient. For this, a minimum of 10 homogeneous negative control and 10 homogeneous positive control (at STC) samples shall be analysed under intermediate precision conditions. The positive control samples shall all be above the cut-off value. In case this criterion is not met, a full validation is required.

4.2.2.5.   Verification of methods already validated through collaborative trials

For screening methods that have already been successfully validated through a collaborative laboratory trial, the method performance shall be verified. For this a minimum of 6 negative control and 6 positive control (at STC) samples shall be analysed. The positive control samples shall all be above the cut-off value. In case this criterion is not met, the laboratory has to perform a root-cause analysis to identify why it cannot meet the specification as obtained in the collaborative trial. Only after taking corrective action, it shall re-verify the method performance in its laboratory. In case the laboratory is not capable to verify the results from the collaborative trial, it will need to determine its own cut-off value in a complete single laboratory validation.

4.2.2.6.   Continuous method verification/on-going method validation

After initial validation, additional validation data are acquired by including at least two positive control samples in each batch of samples screened. One positive control sample shall be a known sample (e.g. one used during initial validation), the other shall be a different commodity from the same commodity group (in case only one commodity is analysed, a different sample of that commodity is used instead). Inclusion of a negative control sample is optional. The results obtained for the two positive control samples are added to the existing validation set.

At least once a year the cut-off value is re-determined and the validity of the method is re-assessed (re-evaluation of the available QA/QC data obtained in the last year). The continuous method verification serves several purposes, including:

• — quality control for the batch of samples screened;

• — providing information on robustness of the method at conditions in the laboratory that applies the method;

• — justification of applicability of the method to different commodities;

• — allowing to adjust cut-off values in case of gradual drifts over time.

4.2.2.7.   Validation report

The validation report shall contain:

• — a statement on the STC;

• — a statement on the determined cut-off value; Note: The cut-off value shall have the same number of significant figures as the STC. Numerical values used to calculate the cut-off value need at least one more significant figure than the STC.

• — a statement on calculated false suspected rate;

• — a statement on how the false suspected rate was generated. Note: The statement on the calculated false suspected rate indicates if the method is fit-for-purpose as it indicates the number of blank (or low level contamination) samples that will be subject to verification. Table 2 Commodity groups for the validation of confirmatory and screening methods Commodity groups Commodity categories Typical representative commodities included in the category High water content Fruit Juices Alcoholic beverages Root and tuber vegetables Cereal or fruit based purees Apple juice, grape juice Wine, beer, cider Fresh ginger, herbal infusions (liquid) Purees intended for infants and small children High oil content Tree nuts Oil seeds and products thereof Oily fruits and products thereof Walnuts, hazelnuts, chestnuts rapeseed, sunflower, cottonseeds, soybeans, peanuts, sesame seeds etc. Oils and pastes (e.g. peanut butter, tahina) High starch and/or protein content and low water and fat content Cereal grain and products thereof Dietary products Wheat, rye, barley, maize, rice, oats Wholemeal bread, white bread, crackers, breakfast cereals, pasta Dried powders for the preparation of food for infants and small children High acid content and high water content [^E0025] Citrus products ‘Difficult or unique commodities’ [^E0026]

Cocoa beans and products thereof, copra and products thereof, coffee, tea (dried product)

Spices, liquorice root, herbal infusions (dried product), food supplements, pollen, and pollen products High sugar low water content Dried fruits Figs, raisins, currants, sultanas Milk and milk products Milk Cheese Dairy products (e.g. milk powder) Cow, goat and buffalo milk Cow, goat cheese Yogurt, cream Meat (tissue) Edible offals Muscle, processed meat products Kidney, liver ham (¹) If a buffer is used to stabilise the pH changes in the extraction step, then this commodity group can be merged into one commodity group ‘High water content’. (2) ‘Difficult or unique commodities’ needs only to be fully validated if they are frequently analysed. If they are only analysed occasionally, validation may be reduced to just checking the reporting levels using spiked blank extracts. Table 3 One tailed t-value for a false negative rate of 5 % Degrees of Freedom Number of replicates t-value (5 %) 10 11 1,812 11 12 1,796 12 13 1,782 13 14 1,771 14 15 1,761 15 16 1,753 16 17 1,746 17 18 1,74 18 19 1,734 19 20 1,729 20 21 1,725 21 22 1,721 22 23 1,717 23 24 1,714 24 25 1,711 25 26 1,708 26 27 1,706 27 28 1,703 28 29 1,701 29 30 1,699 30 31 1,697 40 41 1,684 60 61 1,671 120 121 1,658 ∞ ∞ 1,645

4.2.3.   Requirements for qualitative screening methods (methods that do not give numerical values)

The development of validation guidelines for binary test methods is currently carried out by various standardisation bodies (e.g. AOAC, ISO). AOAC has drafted a guideline on the validation of binary test methods. This document can be regarded as the current state of the art in the field of validation of binary test methods. Therefore, methods that give binary results (e.g. visual inspection of dip-stick tests) should be validated according to AOAC International Guidelines for Validation of Qualitative Binary Chemistry Methods ([^E0027]).

However, other recognised validation guidelines can be used such as the approach provided for in ISO/TS 23758:2021 | IDF/RM 251 Guidelines for the validation of qualitative screening methods for the detection of residues of veterinary drugs in milk and milk products.

4.2.4.   Quantitative determination of ergot sclerotia

Ergot sclerotia in cereals shall be determined by visual (macroscopic/microscopic) identification of the ergot sclerotia and ergot sclerotia fragments. Quantification shall be done by weighing the amount of identified ergot sclerotia and ergot sclerotia fragments with a particle size > 0,5 mm.

4.3.   Estimation of measurement uncertainty, recovery calculation and reporting of results ([^E0028])

4.3.1.   Confirmatory methods

The analytical result shall be reported as follows:

• (a) Corrected for recovery, where appropriate and relevant, and when corrected it shall be stated. The recovery rate is to be quoted unless intrinsic correction for bias is part of the procedure. The correction for recovery is not necessary in case the recovery rate is between 90-110 %.

• (b) As x +/– U whereby x is the analytical result and U is the expanded analytical measurement uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %.

As a possibility a default expanded measurement uncertainty of 50 % may be reported, provided that the laboratory meets all precision requirements specified in point 4.2. An individual laboratory can demonstrate that by achieving the criteria for the repeatability (RSDr) and the within-laboratory reproducibility (RSDwR), supplemented by successful participation in proficiency testing programs (unless no suitable proficiency testing program is available), as a mean z-score of |z| ≤ 2 demonstrates that the required reproducibility (RSDR) is met (based on a target standard deviation of 25 %).

In case the maximum level has been set for the sum of toxins (e.g. aflatoxins, T-2/HT-2-toxin, fumonisins, ergot alkaloids), the analytical results of all individual toxins shall be reported. For ergot alkaloids, it is also allowed to report the sum of each of the six epimer pairs instead of the 12 individual epimers.

Recovery correction, if applicable, shall be done for each of the individual toxins before summation of the concentrations. For ergot alkaloids, the correction can also be done based on the recovery obtained for each of the epimer pairs.

For compliance verification with the sum-ML, a lower-bound approach shall be applied which means that results for individual toxins that are <LOQ shall be replaced by zero for the calculation of the sum.

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 defense or referee purposes, the national rules apply. In particular, if

the analytical result of the official control sample indicates a non-compliance beyond reasonable doubt, taking into account the expanded measurement uncertainty and

the analytical result of the defense sample indicates a non-compliance but not beyond reasonable doubt with a larger expanded measurement uncertainty than the one of the official control,

then the analytical result of the defense sample cannot supersede the non-compliance established for the official control sample.

4.3.2.   Screening methods

The result of the screening shall be expressed as compliant or suspected to be non-compliant.

‘Suspected to be non-compliant’ means the sample exceeds the cut-off value and may contain the mycotoxin at a level higher than the STC. Any suspect result triggers a confirmatory analysis for unambiguous identification and quantification of the mycotoxin.

‘Compliant’ means that the mycotoxin content in the sample is < STC with a level of confidence of 95 % (i.e. there is a 5 % chance that samples will be incorrectly reported as negative). The analytical result is reported as ‘< level of STC’ with the level of STC specified.

4.4.   Laboratory quality standards

A laboratory shall comply with the provisions of Article 37(4) and (5) of Regulation (EU) 2017/625.

[^E0001]: For risk assessment, fit-for-purpose LOQs are generally lower compared to what is required for official control for checking compliance with a ML, as the aim is to generate numerical data for the major part of the samples analysed (i.e. avoid left-censored data) in order to be able to perform accurate exposure assessments. For monitoring purposes, it can be acceptable to report levels below the LOQ as defined in the context of this Regulation.

[^E0002]: Commission Regulation (EC) No 333/2007 of 28 March 2007 laying down the methods of sampling and analysis for the control of the levels of trace elements and processing contaminants in foodstuffs (OJ L 88, 29.3.2007, p. 29).

[^E0003]: A guidance document for competent authorities for the control of compliance with EU legislation on aflatoxins is available at https://food.ec.europa.eu/document/download/5e7138d9-26c5-4f38-900c-9933fe605a92_en?filename=cs_contaminants_sampling_analysis-guidance-2010_en.pdf The guidance document provides additional practical information but the information contained in the guidance document is subordinate to the provisions in this Regulation.

[^E0005]: The sampling of such lots shall be performed in accordance with the rules set out in part N. Guidance for sampling large lots shall be provided in a guidance document available on the following website: https://food.ec.europa.eu/system/files/2016-10/cs_contaminants_sampling_guidance-sampling-final_en.pdf

[^E0007]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0009]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0011]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0013]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0015]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 0,5 kg, the aggregate sample weight might be less than 0,5 kg.

[^E0016]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0018]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0019]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 litre, the aggregate sample volume might be less than 1 litre.

[^E0020]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0021]: In case the portion to be sampled is so small that it is impossible to obtain an aggregate sample of 1 kg, the aggregate sample weight might be less than 1 kg.

[^E0024]: available at: https://food.ec.europa.eu/document/download/f16cac78-9318-4f1f-b2fa-efb25d2f1880_en

[^E0027]: Available at: https://academic.oup.com/jaoac/article-pdf/97/5/1492/32425003/jaoac1492.pdf

[^E0028]: 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’

https://food.ec.europa.eu/system/files/2016-10/cs_contaminants_sampling_analysis-report_2004_en.pdf