COMMISSION REGUL ATION (EU) No 519/2014 
of 16 May 2014 
amending Regulation (EC) No 401/2006 as regards methods of sampling of large lots, spices and 
food supplements, perfor mance criter ia for T-2, HT-2 toxin and citrinin and screening methods of 
analysis 
(Text with EEA relevance) 
THE EUR OPEAN COMMISSION, 
Having regar d to the Treaty on the Functioning of the European Union, 
Having regard to Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 Apr il 2004 on 
official controls perf ormed to ensure the verification of compliance with feed and food law, animal health and animal 
welf are rules (1), in particular Article 11(4) thereof, 
Whereas: 
(1)  Commission Regulation (EC) No 1881/2006 (2) provides for maximum limits for certain mycoto xins in certain 
foodstuffs. 
(2) Sam pling plays a crucial part in the precision of the determination of the levels of mycotoxins, which are hetero ­
geneously distr ibut ed in a lot. It is theref ore necessar y to set out criteria which the sampling methods should 
fulfil. 
(3)  Commission Regulation (EC) No 401/2006 (3) establishes the criteria for the sampling for the control of the 
levels of mycotoxins. 
(4)  It is necessar y to amend the rules concer ning the sampli ng of spices in order to take into account the differ ences 
in particle size which leads to the heterogeneous distr ibution of mycotoxin contamination in spices. Further more 
it is appropr iate to establish rules for the sampli ng of large lots in order to ensure a unif orm enforcement 
approac h across the Union. It is also appropr iate to clarify which method of sampli ng has to be applied for the 
samp ling of apple juice. 
(5)  The performance criteria for T-2 and HT-2 toxin need to be updat ed in order to take into account scientifi c and 
technological progress. Performance criteria for citrinin need to be established given the maximum level estab ­
lished for citrinin in food supplements based on rice fermented with the red yeast Monascus purpureu s. 
(6)  For the analysis of mycotoxins, screening methodologies are used more and more. It is appropr iate to establish 
criteria with whic h the screening methods have to compl y with for use for regulatory purposes. 
(7)  The measures provided for in this Regulation are in accordance with the opinion of the Standing Committ ee on 
the Food Chain and Animal Health, 
HAS ADOPTED THIS REGUL ATION: 
Article 1 
Regulation (EC) No 401/2006 is amended as follows: 
(1)  Annex I is amended as follows: 
(a)  In part B, footnot e (1) is replaced by the following: 
‘(1)  The sampli ng of such lots shall be perf ormed in accordance with the rules set out in part L. Guidance for 
sampling large lots shall be provided in a guidance document available on the following website: http://ec. 
europa.eu/f ood/f ood/chem icalsafety/contaminants/guidance-sam pling-f inal.pdf 17.5.2014 L 147/29 Official Jour nal of the European Union EN     
(1)OJ L 165, 30.4.2004, p. 1. 
(2)Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs 
(OJ L 364, 20.12.2006, p. 5). 
(3)Commission Regulation (EC) No 401/2006 of 23 Febr uary 2006 laying down the methods of samplin g and analysis for the official 
control of the levels of mycoto xins in foodstuffs (OJ L 70, 9.3.2006, p. 12). The application of sampli ng rules in accordance with EN ISO 24333:2009 or GAFT A Sampli ng Rules 124, 
applied by food business operators to ensure compl iance with provisions in legislation is equivalent to the 
sampling rules set out in part L. 
For the sampli ng of lots for Fusar ium-to xins, the application of sampling rules in accordance with EN ISO 
24333:2009 or GAFT A Sampling Rules 124, applied by food business operators to ensure compl iance with 
provisions in legislation is equivalent to the sampli ng rules set out in part B;’ 
(b)  In part B.2, Table 1 is replaced by the following table: 
‘Table 1 
Subdivision of lots into sublots depending on product and lot weight  
Commodity Lot weight (tonne) Weight or number of 
sublots No incremental 
samples Aggrega te sample 
Weight (kg) 
Cereals and cereal 
products > 300 and < 1 500 3 sublots 100 10 
≥ 50 and ≤ 300 100 tonnes 100 10 
< 50 — 3-100 (*) 1-10 
(*)  Depending on the lot weight — see Table 2.’  
(c)  In part B.3, the following senten ce is added at the end of the first indent: 
‘For lots > 500 tonnes, the number of incremental samples is provided for in part L.2 of Annex I.’ 
(d)  In part D.2 the following senten ce is added after the first senten ce: 
‘This method of sampli ng is of also of application for the official control of the maximum levels established for 
ochrat oxin A, aflatoxin B1 and total aflatoxins in spices with a relatively large particle size (par ticle size compar ­
able with peanuts or larger e.g. nutmeg).’; 
(e)  In part E, the first senten ce is replaced by the following: 
‘This method of sampli ng is of application for the official control of the maximum levels established for ochra ­
toxin A, aflatoxin B1 and total aflatoxins in spices excep t in cases of spices with a relatively large particle size 
(heterog eneous distr ibution of mycoto xin contamination).’; 
(f)  In part I, the heading and the first sente nce are replaced by the following: 
‘I.  METHOD OF SAMPLING FOR SOLID APPLE PRODUCTS 
This method of sampling is of application for the official control of the maximum levels established for 
patulin in solid apple products, including solid apple products for infants and young children.’ 
(g)  In part I.1, second paragraph, the following sente nces are deleted: 
‘In case of liquid products the lot shall be thoroughly mixed insofar as possible by either manual or mechanical 
means immediately prior to sampling. In this case, a homog enous distr ibution of patulin can be assumed within 
a given lot. It is theref ore sufficient to take three incremental sam ples from a lot to form the aggregat e sam ple.’ 
(h)  New Parts L and M as set out in Annex I to this Regulation, are added. 
(2) In Annex II, points 4.2 ‘General requirements’, 4.3 ‘Specif ic requirements’ and 4.4 ‘Estimation of measurement uncer ­
tainty , reco very calculation and repor ting of results’ are replaced by the text set out in Annex II to this Regulation. 17.5.2014 L 147/30 Official Jour nal of the European Union EN     Article 2 
This Regulation shall enter into force on the twentieth day following that of its publication in the Official Journal of the 
European Union. 
It shall apply from 1 July 2014. 
This Regulation shall be binding in its entirety and directly applicable in all Member State s. 
Done at Brussels, 16 May 2014. 
For the Commission 
The Presid ent 
José Manuel BARR OSO  17.5.2014 L 147/31 Official Jour nal of the European Union EN     ANNEX I 
‘L.  METHOD OF SAMPLI NG FOR VER Y LARGE LOTS OR LOTS STORED OR TRANSPORTED IN A WAY WHEREBY 
SAMPLING THR OUGHOUT THE LOT IS NOT FEASIBLE 
L.1.  General principles 
In case the way of transpor t or storag e of a lot does not enable to take incremental sam ples throughout the 
whole lot, sampling of such lots should preferably be done when the lot is in flow (dynamic sampli ng). 
In the case of large warehouses destined to store food, operators should be encouraged to install equipment in 
the warehouse enabling (auto matic) sampling across the whole stored lot. 
When the sam pling procedures as provided for in this part L are applied, the food business operat or or his 
representative should be informed of the samp ling procedure. If the sampli ng procedure is questioned by the 
food business operator or his representative, the food business operator or his representative shall enable the 
compet ent author ity to sample throughout the whole lot at his/her own cost. 
Sampli ng of a part of the lot is allowed, on the condition that the quantity of the samp led part is at least 10 % 
of the lot to be sampled. If a part of a lot of food of the same class or descr iption has been sampled and identi ­
fied 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 unsatisfa ctor y. 
The relevant 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 transpor ted in a way whereby 
sampli ng throughout the lot is not feasible. 
L.2.  Number of incrementa l 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 sam ples to 
be taken = 100 incremental samples + √tonn es. 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 B and on the condition that the sublots can be 
separated physically , the number of incremental samples as provided for in part B have to be taken. 
L.3.  Large lots transpor ted by ship 
L.3.1.  Dynamic sampling of large lots transpor ted by ship 
The sam pling of large lots in ships is preferably carried out while the product is in flow (dynamic sam pling). 
The sampling is to be done per hold (entity that can physically be separated ). Holds are however emp tied partly 
one after the other so that the initial physical separation no long er exists after transfer into storag e facilities. 
Sampli ng can theref ore be performe d 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. Nor mally , sampli ng has to be performed at regular inter vals 
during the whole duration of unloading. It is however not alwa ys feasible or appropr iate for an offici al 
inspector to be present for sampli ng during the whole operation of unloading. Theref ore sampling of part of 
the lot is allowed to be under taken (sampled portion). The number of incremental samples is determi ned by 
taking into account the size of the sam pled portion. 
Even if the offici al sample is taken autom atically , the presence of an inspect or is necessar y. However if the auto ­
matic sampling is done with pre-set parameter s which cannot be changed during the sam pling and the incre ­
mental sam ples are collected in a sealed receptacle, preventing any possible fraud, then the presence of an 
inspector is only required at the beginning of the sampli ng, ever y time the recep tacle of the sam ples needs to 
be change d and at the end of the sam pling. 
L.3.2.  Sampling of lots transpor ted by ship by static sampling 
In cases where the sampli ng is done in a static way the same procedure as foreseen for storage facilities (silos) 
accessible from above has to be applied (see point L.5.1). 
The sampli ng has to be performe d on the accessible part (from above) of the lot/hold. The number of incre ­
mental sam ples is determined by taking into account the size of the sample d portion. 17.5.2014 L 147/32 Official Jour nal of the European Union EN     L.4.  Sampling of large lots stored in warehouses 
The sampli ng has to be performe d on the accessible part of the lot. The number of incremental samples is 
determi ned by taking into account the size of the sampled portion. 
L.5.  Sampling of storage facilities (silos) 
L.5.1.  Sampling of silos (easily) accessible from above 
The sampli ng has to be performe d on the accessible part of the lot. The number of incremental samples is 
determi ned by taking into account the size of the sampled portion. 
L.5.2.  Sampling of silos not accessible from above (closed silos) 
L.5.2.1.  Silos not accessible from abo ve (closed silos) with individual sizes > 100 tonnes 
Food stored in such silos cannot be sam pled in a static way. Theref ore when the food in the silo has to be 
sampled and there is no possibility to move the consignment, the agreement has to be made with the operat or 
that he or she has to inform the inspect or about when the silo will be unloaded, partially or compl etely , in 
order to enable sampling when the food is in flow. 
L.5.2.2.  Silos not accessible from abo ve (closed silos) with individual sizes < 100 tonnes 
Contrar y to the provision in part point L.1 (sampled part at least 10 %), the sam pling procedure involves the 
release into a receptacle of a quantity of 50 to 100 kg and taking the sam ple from it. The size of the aggregat e 
sample corresponds to the whole lot and the number of incremental sample s relat e to the quantity of the food 
from the silo released into the receptacle for sampling. 
L.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 
impor t or control and theref ore the sampli ng should take place when such containers are unloaded. The 
operat or has to inform the inspect or about the place and time of unloading the containers. 
M.  METHOD OF SAMPLING OF FOOD SUPPLEMENTS BASED ON RICE FERMENTED WITH RED YEAST MONASCUS 
PURPUREUS 
This method of sampling is applicable to the official control of the maximum level established for citrinin in 
food supplements based on rice fermented with red yeast Monascus purpureus. 
Sampling procedure and sample size 
The samp ling procedure is on the supposition that the food supplements based on rice fermented with red 
yeast Monascus purpureus are market ed in retail packag es containing usually 30 to 120 capsules per retail 
packag e. 
Lot size (number of retail 
packages) Number of retail packages 
to be taken for sample Sample size 
1-50 1 All capsules 
51-250 2 All capsules 
251-1 000 4 From each retail packag e taken for sample, half of the 
capsules 
> 1 000 4 + 1 retail packag e per 
1 000 retail packag es 
with a maximum of 25 
retail packag es ≤ 10 retail packag es: from each retail packag e, half of 
the capsules 
> 10 retail packag es: from each retail packag e, an equal 
number of capsules is taken to result in a sample with 
the equivalent of the content of retail 5 packa ges’   17.5.2014 L 147/33 Official Jour nal of the European Union EN     ANNEX II 
‘4.2.  General requirements 
Confir matory methods of analysis used for food control purposes shall compl y with the provisions of 
items 1 and 2 of Annex III to Regulation (EC) No 882/2004. 
4.3.  Specif ic requirements 
4.3.1.  Specific requir ements for confir mator y methods 
4.3.1.1.  Perf ormance criteria 
It is recommended that fully validat ed conf irmatory methods (i.e. methods validated by collaborative trials for 
relevant matr ices) are used where appropr iate and available. Other suitable validat ed conf irmatory methods 
(e.g. methods validated in-house on relevant matr ices belonging to the commodity group of inter est) may 
also be used provided they fulfil the perf ormance criteria set out in the following tables. 
Where possible, the validation of in-house validat ed methods shall include a certified reference mater ial. 
(a)  Performanc e criteria for aflatoxins 
Criterion Concentration Range Recommended Value Maximum permitt ed Value 
Blanks All Neg ligible —     
Reco very — Aflatoxin 
M1 0,01-0,05 mg/kg 60 to 120 %   
> 0,05 mg/kg 70 to 110 %      
Reco very-Af latoxins B1, 
B2, G1, G2 < 1,0 mg/kg 50 to 120 %   
1-10 mg/kg 70 to 110 %   
> 10 mg/kg 80 to 110 %      
Reproducibility RSDR All As derived from 
Hor witz Equation (*)(**) 2 × value derived from 
Hor witz Equation (*)(**) 
Repeatability RSDr may be calculat ed as 0,66 times Reproducibility RSDR at the concentration of interest.   
Note: 
—  Values to apply to both B1 and sum of B1 + B2 + G1 + G2 
—  If sum of individual aflatoxins B1 + B2 + G1 + G2 are to be repor ted, then response of each to the 
analytical syste m must be either known or equivalent.  
(b)  Performanc e criteria for ochrat oxin A 
Level 
µg/kg Ochrat oxin A 
RSDr % RSDR % Reco very % 
< 1 ≤ 40 ≤ 60 50 to 120 
≥ 1 ≤ 20 ≤ 30 70 to 110   17.5.2014 L 147/34 Official Jour nal of the European Union EN     (c)  Performanc e criteria for patulin 
Level 
µg/kg Patulin 
RSDr % RSDR % Reco very % 
< 20 ≤ 30 ≤ 40 50 to 120 
20-50 ≤ 20 ≤ 30 70 to 105 
> 50 ≤ 15 ≤ 25 75 to 105   
(d)  Performanc e criteria for deoxynivalenol 
Level 
µg/kg Deo xynivalenol 
RSDr % RSDR % Reco very % 
> 100-≤ 500 ≤ 20 ≤ 40 60 to 110 
> 500 ≤ 20 ≤ 40 70 to 120   
(e)  Performanc e criteria for zearalenone 
Level 
µg/kg Zearalenone 
RSDr % RSDR % Reco very % 
≤ 50 ≤ 40 ≤ 50 60 to 120 
> 50 ≤ 25 ≤ 40 70 to 120   
(f)  Performanc e criteria for Fumonisin B1 and B2 individually 
Level 
µg/kg Fumonisin B1 and B2 individually 
RSDr % RSDR % Reco very % 
≤ 500 ≤ 30 ≤ 60 60 to 120 
> 500 ≤ 20 ≤ 30 70 to 110   
(g)  Performanc e criteria for T-2 and HT-2 toxin individually 
Level 
µg/kg T-2 and HT-2 toxin individually 
RSDr % RSDR % Reco very % 
15-250 ≤ 30 ≤ 50 60 to 130 
> 250 ≤ 25 ≤ 40 60 to 130   
(h)  Performanc e criteria for citrinin 
Level 
µg/kg Citrinin 
RSDr % Recommended RSDR % Maximum allowed RSDR % Reco very % 
All 0,66 × RSDR As derived from Hor witz 
Equation (*)(**) 2 × value derived from 
Hor witz Equation (*)(**) 70 to 120   17.5.2014 L 147/35 Official Jour nal of the European Union EN     (i)  Notes to the perf ormance criteria for the mycotoxins: 
—The detection limits of the methods used are not state d as the precision values given at the concentra ­
tions of inter est. 
—  The precision values are calculat ed from the Hor witz equation, in particular the original Hor witz 
equation (for concentrations 1,2 × 10–7 ≤ C ≤ 0,138) (*) and the modified Hor witz equation (for 
concentrations C < 1,2 × 10–7) (**). 
(*)  Hor witz equation for concentrations 1,2 × 10–7 ≤ C ≤ 0,138: 
RSDR = 2(1-0.5logC) 
(ref: W. Horwitz, L.R. Kamps, K.W . Boyer , J.Assoc.Of f.Analy .Chem.,1980, 63, 1344) 
(**)  Modifi ed Hor witz equation (*) for concentrations C < 1,2 × 10–7: 
RSDR = 22 % 
(ref: M. Thompson, Analys t, 2000, 125, p. 385-386) 
Where: 
—  RSDR is the relative standard deviation calculat ed from results generate d under reproducibility 
conditions [(sR/) × 100] 
—  C is the concentration ratio (i.e. 1 = 100g/100g, 0,001 = 1 000 mg/kg) 
This is a general ised precision equation which has been found to be independent of analyt e and 
matr ix but solely dependent on concentration for most routine methods of analysis. 
4.3.1.2.  “Fitness-f or-pur pose” approach 
For in-house validat ed methods, as an alternative, a “fitness-f or-pur pose” approach (***) may be used to assess 
their suitability for official control. Methods suitable for official control must produce results with a standard 
measurement uncer tainty (u) less than the maximum standard measurement uncer tainty calculated using the 
formula belo w: 
Uf¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi
ðLOD=2Þ2þðα�CÞ2q
where: 
—  Uf is the maximum standard measurement uncer tainty (µg/kg) 
—  LOD is the limit of detection of the method (µg/kg) 
—  α is a constant, numer ic factor to be used depending on the value of C. The values to be used are set out 
in Table hereaf ter. 
—  C is the concentration of interest (µg/kg) 
If the analytical method provides results with uncer tainty measurements less than the maximum standard 
uncer tainty the method shall be considered being equally suitable to one whic h meets the performa nce 
criteria given in point 4.3.1.1. 
Table 
Numer ic values to be used for α as const ant in formula set out in this point, depending on the 
concentration of interes t 
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  
(***) Ref: M. Thompson and R. Wood, Accred. Qual. Assur ., 2006, 10, p. 471-478.  17.5.2014 L 147/36 Official Jour nal of the European Union EN     4.3.2.  Specific requir ements for semi-quantitative screening methods 
4.3.2.1.  Scope 
The scope applies to bioanalytical methods based on immuno-recognition or recept or binding (such as 
ELISA, dip-sticks, lateral flow devices, immuno-sensors) and physicoc hemical methods based on chromato ­
graphy or direct detection by mass spectrometr y (e.g. ambient MS). Other methods (e.g. thin layer chromato ­
graphy) are not excl uded provided the signals generat ed relat e directly to the mycoto xins of inter est and allow 
that the principle descr ibed hereunder is applicable. 
The specifi c requirements apply to methods of which the result of the measurement is a numer ical 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 numer ical values (e.g. only a line that is present 
or absent), whic h require differ ent validation approaches. Specific requirements for these methods are 
provided in point 4.3.3. 
This document descr ibes procedures for the validation of screening methods by means of an inter -laboratory 
validation, the verification of the performa nce of a method validat ed by means of an inter -laboratory exercise 
and the sing le-laboratory validation of a screening method. 
4.3.2.2.  Terminology 
Screening target concentration (STC): the concentration of interest for detection of the mycoto xin in a 
sample . When the aim is to test compliance with regulatory limits, the STC is equal to the applicable 
maximum level. For other purposes or in case no maximum level has been established, the STC is predef ined 
by the laboratory . 
Screening method: means method used for selection of those samples with levels of mycotoxins that excee d 
the screening target concentration (STC), with a given certainty . For the purpose of mycoto xin screening, a 
certainty of 95 % is considered fit-for-pur pose. The result of the screening analysis is either “negative” or 
“suspect”. Screening methods shall allow a cost-eff ective high sample-throughput, thus increasing the chance 
to disco ver new incidents with high exposure and health risks to consumers. These methods shall be based 
on bio-analytical, LC-MS or HPLC methods. Results from samples exceed ing the cut-off value shall be verified 
by a full re-analysis from the original sample by a conf irmatory method. 
“Nega tive sample” means the mycoto xin conte nt in the sample is < STC with a certainty of 95 % (i.e. there is 
a 5 % chance that sam ples will be incor rectly repor ted as nega tive). 
“False negat ive sample” means the mycotoxin conte nt in the samp le is > STC but it has been identif ied as 
negat ive. 
“Suspect sample” (screen positive) means the samp le exceed s the cut-off level (see belo w) and may contain 
the mycoto xin at a level higher than the STC. Any suspect result triggers a conf irmatory analysis for unam ­
biguous identif ication and quantifi cation of the mycotoxin. 
“False suspect sam ple” is a negative sample that has been identif ied as suspect. 
“Confir mat ory methods” means methods that provide full or compl ementar y information enabling the myco­
toxin to be identif ied and quantifi ed unequivocally at the level of interest. 
Cut-off level: the response, signal, or concentration, obtained with the screening method, above which the 
sample is classified as “suspect”. The cut-off is determined during the validation and take s the variability of 
the measurement into account. 17.5.2014 L 147/37 Official Jour nal of the European Union EN     Neg ative control (blank matr ix) sample: a samp le known to be free (1) of the mycoto xin to be screened for, 
e.g. by previous deter mination using a conf irmatory method of suffi cient sensitivity . If no blank samples can 
be obtained, then mater ial with the lowest obtainable level might be used as long as the level allows the 
conclusion that the screening method is fit for purpose. 
Positive control sample: samp le containing the mycoto xin at the screening targe t concentration, e.g. a certified 
reference mater ial, a mat erial of known content (e.g. test mate rial of prof iciency tests) or other wise suffi ­
ciently charact erised by a confir matory method. In the absence of any of the above, a blend of samples with 
differ ent levels of contamination or a spik ed sam ple prepared within laboratory and suffi ciently charact erised 
can be used, provided it can be proven that the contamination level has been verified. 
4.3.2.3.  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 deter mination of the false negat ive and false suspect rate. In these two 
parameter s performance charact eristics such as sensitivity , selectivity , and precision are embedded. 
Screening methods can be validate d by inter -laboratory or by sing le laborat ory validation. If inter-laboratory 
validation data is already available for a certain mycotoxin/matr ix/STC combination, a verification of method 
performa nce is sufficient in a laboratory imp lementing the method. 
4.3.2.3.1.  Initial validation by sing le laboratory validation 
Mycotoxins: 
The validation shall be performed for ever y 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; fumo ­
nisins B1 & B2), applicability must be demonstrated and limitations of the test mentioned in the scope of the 
method. Undesired cross-reactivity (e.g. DON-3-g lycoside, 3- or 15-acetyl-DON for immuno-based methods 
for DON) is not considered to increase the false negativ e rate of the targe t mycotoxins, but may increase the 
false suspect rate. This unwant ed increasing will be diminished by confir matory analysis for unambiguous 
identif ication and quantif ication of the mycotoxins. 
Matr ices: 
An initial validation should be performe d for each commodity , or, when the method is kno wn to be applic ­
able to multiple commodities, for each commodity group. In the latte r case, one representative and relevant 
commodity is selecte d from that group (see table A). 
Samp le set: 
The minimum number of differ ent sam ples required for validation is 20 homogeneous negative control 
sample s and 20 homogeneous positive control sam ples that contain the mycoto xin at the STC, analysed 
under intermediate precision (RSDRi) conditions spread over 5 differ ent days. Opti onally , additional sets of 
20 samples containing the mycotoxin at other levels can be added to the validation set to gain insight to 
what extent the method can distinguish between different mycoto xin concentrations. 
Concentration: 
For each STC to be used in routine application, a validation has to be performe d. 
4.3.2.3.2.  Initial validation through collaborative trials 
Validation through collaborative trials shall be done in accordance with an intern ationally recognised protocol 
on collaborative trials (e.g. ISO 5725:1994 or the IUPAC Internati onal Harmonised Protocol) which requires 
inclusion of valid data from at least eight differ ent laboratori es. Other than that, the only diffe rence compared 
to sing le laboratory validations is that the ≥ 20 samples per commodity/level can be evenly divided over the 
participating laborat ories, with a minimum of two sample s per laboratory . 17.5.2014 L 147/38 Official Jour nal of the European Union EN     
(1)Sampl es are considered free of analyte if the amount present in the sample does not exceed more than 1/5th of the STC. If the level can be 
quantified with a confir matory method, the level must be taken into consideration for the validation assessment. 4.3.2.4.  Det ermination of cut-off level and rate of false suspected results of blank samp les 
The (relative) responses for the negative control and positive control samples are taken as basis for the calcu ­
lation of the required parameters. 
Screening methods with a response propor tional with the mycotoxin concentration 
For screening methods with a response propor tional with the mycoto xin concentration the following applies: 
Cut-of f = 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 B) 
SDSTC =  standard deviationScreening methods with a response inversely propor tional with the mycotoxin 
concentration 
Similarly , for screening methods with a response inve rsely propor tional with the mycoto xin concentration, 
the cut-off is deter mined as: 
Cut-off = RSTC + t-value0,05 * SDSTC 
By using this specif ic t-value for establishing the cut-off value, the rate of false negativ e results is by default 
set at 5 %. 
Fitness for purpose assessment 
Results from the negative control samp les are used to estimat e the corresponding rate of false suspect results. 
The t-value is calculat ed 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 – meanblank)/SDblankfor screening methods with a response propor tional with the mycotoxin 
concentration 
or 
t-value  =(meanblank – cut off)/SDblankfor screening methods with a response inversely propor tional with the 
mycotoxin concentration 
From the obtained t-value, based on the degrees of freedom calculated from the number of exper iments, the 
probability of false suspect sam ples for a one tailed distr ibution can either be calculat ed (e.g.. spread sheet 
function “TDIST”) or taken from a table for t-distr ibution. 
The corresponding value of the one tailed t-distr ibution specif ies the rate of false suspect results. 
This concept is descr ibed in detail with an example in Analytical and Bioanalytical Chemistr y DOI 10.1007/ 
s00216 -013-6922-1. 
4.3.2.5.  Extension of the scope of the method 
4.3.2.5.1.  Exten sion of scope to other mycotoxins: 
When new mycotoxins are added to the scope of an existing screening method, a full validation is required to 
demonstrate the suitability of the method. 
4.3.2.5.2.  Exten sion to other commodities: 
If the screening method is kno wn or expecte d 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 A) for which an initial validation has already been perf ormed, a limited additional validation is suffi ­
cient. For this, a minimum of 10 homog eneous negativ e control and 10 homog eneous positive control (at 
STC) samples shall be analysed under interm ediate precision conditions. The positive control sam ples shall all 
be above the cut-off value. In case this criterion is not met, a full validation is required. 17.5.2014 L 147/39 Official Jour nal of the European Union EN     4.3.2.6.  Verification of methods already validat ed through collaborative trials 
For screening methods that have already been successfully validate d through a collaborative laborat ory trial, 
the method performa nce shall be verified. For this a minimum of 6 negative control and 6 positive control 
(at STC) sam ples 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-ver ify the 
method performa nce in its laborat ory. In case the laboratory is not capable to verify the results from the 
collaborative trial, it will need to establish its own cut-off in a complet e sing le laboratory validation. 
4.3.2.7.  Continuous method ver ification/on-going method validation 
After initial validation, additional validation data are acquired by including at least two positive control 
sample s in each batch of sam ples screened. One positive control sample is a kno wn sample (e.g. one used 
during initial validation), the other is a diffe rent commodity from the same commodity group (in case only 
one commodity is analysed, a differ ent samp le of that commodity is used instead). Inclusion of a nega tive 
control sample is optional. The results obtained for the two positive control sam ples are added to the existing 
validation set. 
At least once a year the cut-off value is re-established and the validity of the method is re-assessed. The 
continuous method verification serves several purposes: 
—  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 diffe rent commodities 
—  allowing to adjust cut-off values in case of gradual drifts over time. 
4.3.2.8.  Validation repor t 
The validation repor t shall contain: 
—  A statement on the STC 
—  A statement on the obtained cut-off. 
Note: The cut-off must have the same number of signif icant figures as the STC. Numer ical values used to 
calculat e the cut-off need at least one more signif icant figure than the STC. 
—  A statement on calculated false suspect ed rate 
—  A statement on how the false suspected rate was generated. 
Note: The statement on the calculat ed false suspect ed rate indicates if the method is fit-for-pur pose as it indi ­
cate s the number of blank (or low level contamination) samples that will be subject to verification. 
Table A 
Commodity groups for the validation of screening methods 
Commodity groups Commodity categor ies Typical representative commodities 
included in the category 
High water conte nt Fruit Juices Apple juice, grape juice 
Alcoholic beverages Wine, beer , cider 
Root and tuber vegetables Fresh ginger 
Cereal or fruit based purees Purees intended for infants and 
small children 17.5.2014 L 147/40 Official Jour nal of the European Union EN     Commodity groups Commodity categor ies Typical representative commodities 
included in the category 
High oil content Tree nuts Walnut, hazelnut, chestnut 
Oil seeds and products thereof Oilseed rape, sunf lower, cotton- 
seed, soybeans, peanuts, sesame 
etc. 
Oily fruits and products thereof Oils and past es (e.g. peanut butter , 
tahina) 
High starc h and/or protein cont ent 
and low water and fat conte nt Cereal grain and products thereof Wheat, rye, barle y, maize, rice, oats 
Wholemeal bread, whit e bread, 
crack ers, breakf ast cereals, pasta 
Dietar y products Dried powders for the preparation 
of food for infants and small chil­
dren 
High acid content and high water 
conte nt(*) Citrus products  
“Difficult or unique commod ­
ities” (**)  Cocoa beans and products thereof, 
copra and products thereof, 
coffee, tea 
Spices, liquor ice 
High sugar low water content Dried fruits Figs, raisins, currants, sultanas 
Milk and milk products Milk Cow, goat and buff alo milk 
Cheese Cow, goat cheese 
Dair y products (e.g. milk powder) Yogur t, cream 
(*)  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”. 
(**)  “Difficult or unique commodities” should only be fully validated if they are frequently analysed. If they are only 
analysed occasionally , validation may be reduced to just checking the repor ting levels using spiked blank extracts.  
Table B 
One tailed t-value for a false negativ e 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 17.5.2014 L 147/41 Official Jour nal of the European Union EN     Degrees of Freedom Number of replicates t-value (5 %) 
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  
4.3.3.  Requirements for qualitativ e screening methods (methods that do not give numer ical values) 
The development of validation guidelines for binar y test methods is currently subject of various standardiza ­
tion bodies (e.g. AOAC, ISO). Very recently AOAC has draf ted a guideline on this matter . This document can 
be regarded as the current state of the art in its field. Theref ore methods that give binar y results (e.g. visual 
inspection of dip-stick tests) should be validated according to this guideline 
http://www .aoac.org/imis15_prod/A OAC_Docs/ISP AM/Qual_Chem_Guideline_Final_Appro ved_031412.pdf 
4.4.  Estimation of measurement uncer tainty , reco very calculation and repor ting of results (1) 
4.4.1.  Confir mator y methods 
The analytical result must be repor ted as follows: 
(a)  Corrected for reco very, the level of reco very being indicated. The correction for reco very is not necessar y 
in case the reco very rate is between 90-110 %. 
(b)  As x +/– U whereby x is the analytical result and U is the expanded measurement uncer tainty , using a 
coverag e factor of 2 whic h gives a level of conf idence of appro ximately 95 %. 
For food of animal origin, the taking into account of the measurement uncer tainty can also be done by estab ­
lishing the decision limit (CCα) in accordance with Commission Decision 2002/657/EC (2) (point 3.1.2.5 of 
Annex I — the case of substances with established permitted limit). 
However if the result of the analysis is significantly (> 50 %) lower than the maximum level or much higher 
than the maximum level (i.e. more than 5 times the maximum level), and on the condition that the appro ­
priate quality procedures are applied and the analysis serves only the purpose of checking compl iance with 
legal provisions, the analytical result might be repor ted without correction for reco very and the repor ting of 
the reco very rate and measurement uncer tainty might be omitt ed in these cases. 17.5.2014 L 147/42 Official Jour nal of the European Union EN     
(1)More details on procedures for the estimation of measurement uncer tainty and on procedures for assessing reco very can be found in the 
repor t “Repor t on the relationship between analytical results, measurement uncer tainty , reco very factors and the provisions of EU food 
and feed legislation” — http://ec.europa.eu/f ood/f ood/chemicalsaf ety/contaminants/repor t-sampl ing_analysis_2004_en.pdf 
(2)Commission Decision 2002/657/EC of 14 Aug ust 2002 implementing Council Directive 96/23/EC concer ning the performance of 
analytical methods and the interpretation of results (OJ L 221, 17.8.2002, p. 8). The present interpr etation rules of the analytical result in view of acceptance or rejection of the lot apply to 
the analytical result obtained on the sam ple for official control. In case of analysis for defence or referee 
purposes, the national rules apply . 
4.4.2.  Screen ing methods 
The result of the screening shall be expressed as compliant or suspect ed to be non-com pliant. 
“Suspecte d to be non-com pliant” means the sample excee ds the cut-off level and may contain the mycotoxin 
at a level higher than the STC. Any suspect result triggers a confir matory analysis for unambiguous identif ica­
tion and quantifi cation of the mycoto xin. 
“Comp liant” means that the mycotoxin conte nt in the sample is < STC with a certainty of 95 % (i.e. there is a 
5 % chance that samples will be incor rectly repor ted as nega tive). The analytical result is repor ted as “< level 
of STC” with the level of STC specified.’  17.5.2014 L 147/43 Official Jour nal of the European Union EN