Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance)

In all other cases, including when no declaration of content has been provided to the laboratory, a second determination shall be carried out from a new sub-sample. If, following the second determination carried out in accordance with the observation flowchart in Diagram 2, or Diagram 3 when appropriate, the sum of the animal particles of a given nature detected over the two determinations is higher than 10, the result of the analysis shall be reported per animal nature using the wording set out in point 2.1.5.3. Otherwise, the result of the analysis shall be reported per animal nature using the wording set out in point 2.1.5.2.

When reporting the results, the laboratory shall indicate on which type of material the analysis has been carried out (sediment, flotate, final flotate or raw material). The reporting shall clearly indicate how many determinations have been carried out and if sieving of the fractions prior to slide preparation, in accordance with point 2.1.3.4.3, first indent, third paragraph, or point 2.1.3.4.4, third indent, was not performed.

The laboratory report shall at least contain information on the presence of constituents derived from terrestrial vertebrates and from fish.

The different situations shall be reported in the following ways.

2.1.5.1.No animal particle of a given nature is detected:

— ‘As far as was discernible using a light microscope, no particle derived from terrestrial vertebrates was detected in the submitted sample.’

— ‘As far as was discernible using a light microscope, no particle derived from fish was detected in the submitted sample.’

— ‘As far as was discernible using a light microscope, no particle derived from terrestrial invertebrates was detected in the submitted sample.’

2.1.5.2.Between 1 and 5 animal particles of a given nature are detected when only one determination has been performed, or between 1 and 10 particles of a given nature are detected in case of two determinations (the number of detected particles is below the decision limit established in the SOP of the EURL-AP and published on its website):

When only one determination has been performed:

— ‘As far as was discernible using a light microscope, no more than 5 particles derived from terrestrial vertebrates were detected in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

— ‘As far as was discernible using a light microscope, no more than 5 particles derived from fish were detected in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

When two determinations have been performed:

— ‘As far as was discernible using a light microscope, no more than 10 particles derived from terrestrial vertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

— ‘As far as was discernible using a light microscope, no more than 10 particles derived from fish were detected over the two determinations in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

— ‘As far as was discernible using a light microscope, no more than 10 particles derived from terrestrial invertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

Additionally:

— In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination.

— When only animal particles which cannot be categorised as either terrestrial vertebrates or fish are detected (e.g. muscle fibres), the report shall mention that only such animal particles were detected and that it cannot be excluded that they originate from terrestrial vertebrates.

2.1.5.3.More than 5 animal particles of a given nature are detected when only one determination has been performed, or more than 10 particles of a given nature are detected in case of two determinations:

When only one determination has been performed:

— ‘As far as was discernible using a light microscope, more than 5 particles derived from terrestrial vertebrates were detected in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)].’

— ‘As far as was discernible using a light microscope, more than 5 particles derived from fish were detected in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)].’

— ‘As far as was discernible using a light microscope, more than 5 particles derived from terrestrial invertebrates were detected in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)].’

When two determinations have been performed:

— ‘As far as was discernible using a light microscope, more than 10 particles derived from terrestrial vertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)].’

— ‘As far as was discernible using a light microscope, more than 10 particles derived from fish were detected over the two determinations in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)].’

— ‘As far as was discernible using a light microscope, more than 10 particles derived from terrestrial invertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)].’

Additionally:

— In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination.

— When only animal particles which cannot be categorised as either terrestrial vertebrates or fish are detected (e.g. muscle fibres), the report shall mention that only such animal particles were detected and that it cannot be excluded that they originate from terrestrial vertebrates.

Deoxyribonucleic acid (DNA) fragments of animal origin which may be present in feed materials and compound feed are detected by a genetic amplification technique through PCR, targeting species-specific DNA sequences.

The PCR method first requires a DNA extraction step. The amplification step shall be applied afterwards to the so-obtained DNA extract, in order to detect the animal species targeted by the assay.

Only reagents approved by the EURL-AP and published on its website shall be used.

Only primers and probes with sequences of oligonucleotides validated by the EURL-AP shall be used (⁴⁸).

Only Master Mix solutions which do not contain reagents susceptible to lead to false results due to presence of animal DNA shall be used (⁴⁹).

2.2.2.2.6. Standard molecular biology plastic-ware: microfuge tubes, filtered plastic tips for micropipettes, plates suitable for the thermocycler.

A representative sample, taken in accordance with the provisions laid down in Annex I, shall be used.

The preparation of laboratory samples up to DNA extraction shall comply with the requirements set out in Annex II. At least 50 g of the sample shall be sub-sampled for analysis and subsequently ground.

The sample preparation shall be performed in a room different from the ones dedicated to DNA extraction and to genetic amplification reactions as described by ISO 24276.

Two test portions of at least 100 mg each shall be prepared.

The DNA extraction shall be performed on each test portion prepared using the SOP established by the EURL-AP and published on its website.

Two extraction controls shall be prepared for each extraction series as described by ISO 24276.

— an extraction blank control,

— a positive DNA extraction control.

The genetic amplification shall be performed using the methods validated for each species requiring identification. These methods are laid down in the SOP established by the EURL-AP and published on its website. Each DNA extract shall be analysed at least at two different dilutions in order to evaluate inhibition.

Two amplification controls shall be prepared per species target as described by ISO 24276.

— a positive DNA target control shall be used for each plate or series of PCR assays,

— an amplification reagent control (also called no template control) shall be used for each plate or series of PCR assays.

When reporting the results, the laboratory shall indicate at least the weight of the test portions used, the extraction technique used, the number of determinations carried-out and the limit of detection of the method.

Results shall not be interpreted and reported if the positive DNA extraction control and the positive DNA target controls do not provide positive results for the target under assay while the amplification reagent control is negative.

In case results from the two test portions are not consistent, at least the genetic amplification step shall be repeated. If the laboratory suspects that the DNA extracts can be the cause of the inconsistency, a new DNA extraction and a subsequent genetic amplification shall be performed before interpreting the results.

The final expression of the results shall be based on the integration and the interpretation of the results of the two test portions in accordance with the SOP established by the EURL-AP and published on its website.

A negative result shall be reported as follows:

No DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay).

A positive result shall be reported as follows:

DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay).

ANNEX VII

METHOD OF CALCULATING THE ENERGY VALUE OF POULTRY FEED

1. METHOD OF CALCULATION AND EXPRESSION OF ENERGY VALUE

The energy value of compound poultry feed must be calculated in accordance with the following formula set out on the basis of the percentages of certain analytical components of the feed. This value is to be expressed in megajoules (MJ) of metabolisable energy (ME), corrected for nitrogen, per kilogram of compound feed:

MJ/kg of ME = 0,1551 × % crude protein + 0,3431 × % crude fat + 0,1669 × % starch + 0,1301 × % total sugar (expressed as sucrose).

2. TOLERANCES APPLICABLE TO DECLARED VALUES

If the official inspection reveals a discrepancy (increased or reduced energy value of the feed) between the result of the inspection and the declared energy value, a tolerance of 0,4 MJ/kg of ME shall be permitted.

3. EXPRESSION OF RESULT

After application of the above formula, the result obtained must be given to one decimal place.

4. SAMPLING AND ANALYSIS METHODS

Sampling of the compound feed and determination of the content of analytical components indicated in the method of calculation must be performed in accordance with the Union sampling methods and analysis methods for the official control of feed respectively.

The following are to be applied:

— for determining the crude fat content: procedure B of the method for the determination of crude oils and fats, laid down in Part G of Annex III,

— for determining the starch content: the polarimetric method, laid down in Part K of Annex III

METHOD OF CALCULATING THE ENERGY VALUE IN FEED MATERIALS AND COMPOUND FEED FOR CATS AND DOGS

The energy value in feed materials and compound feed for cats and dogs is to be calculated in accordance with EN 16967 Animal feeding stuffs: Methods of sampling and analysis – Predictive equations for metabolisable energy in feed materials and compound feed (pet food) for cats and dogs including dietetic food.

ANNEX IX

1. Directive 71/250/EEC

2. Directive 71/393/EEC

3. Directive 72/199/EEC

4. Directive 73/46/EEC

5. Directive 76/371/EEC

6. Directive 76/372/EEC

7. Directive 78/633/EEC

8. Directive 81/715/EEC

9. Directive 84/425/EEC

10. Directive 86/174/EEC

11. Directive 93/70/EEC

12. Directive 93/117/EC

13. Directive 98/64/EC

14. Directive 1999/27/EC

15. Directive 1999/76/EC

16. Directive 2000/45/EC

17. Directive 2002/70/EC

18. Directive 2003/126/EC