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4.11.2011 Official
Journal
of
the
European
Union L
287/23
COMMISSION IMPLEMENTING REGULATION (EU) No 1109/2011
of 3 November 2011
amending Annex I to Regulation (EC) No 2075/2005 as regards the equivalent methods for
Trichinella testing
(Text with EEA relevance)
THE EUROPEAN COMMISSION,
apparatuses for testing of Trichinella by the digestion
method were endorsed unanimously during the meeting
Having regard to the Treaty on the Functioning of the European
of the Standing Committee on the Food Chain and
Union,
Animal Health on 16 December 2008.
Having regard to Regulation (EC) No 854/2004 of the European
(4) In
2010
a
new
apparatus
method
for
testing
of Trichinella
Parliament and of the Council of 29 April 2004 laying down
in domestic swine was validated by the EU Reference
specific rules for the organisation of official controls on
Laboratory for parasites in accordance with those
products of animal origin intended for human consumption (
guidelines.

1 ),
and in particular the former part of the introductory phrase of
Article 18 and points 8, 9 and 10 of that Article,
(5)
Results of the validation show that the new apparatus
and related method of detection of Trichinella, validated
Whereas:
under the code of the EU Reference Laboratory, No
EURLP_D_001/2011 ( 4 ), is equivalent to the reference
method as laid down in Chapter I of Annex I to Regu
(1) Commission
Regulation
(EC)
No
2075/2005 of
lation (EC) No 2075/2005. Therefore, it should be
5 December 2005 laying down specific rules on official
included in the list of equivalent methods of detection
controls for Trichinella in meat ( 2 ) provides for methods
listed in Chapter II of Annex I to Regulation (EC) No
of detection of Trichinella in samples of carcases. The
2075/2005.
reference method is laid down in Chapter I of Annex I
to that Regulation. Three methods of detection equivalent
to the reference method are laid down in Chapter II of
(6) Chapter
II
of
Annex
I
to
Regulation
(EC)
No
2075/2005
Annex I to that Regulation.
should therefore be amended accordingly.


(7) The
measures
provided
for
in
this
Regulation
are
in
(2) Regulation
(EC)
No
2075/2005,
as
amended
by
Regu
accordance with the opinion of the Standing
lation (EC) No 1245/2007 ( 3 ), permits the use of liquid
Committee on the Food Chain and Animal Health,
pepsin for the detection of Trichinella in meat and estab

lishes its requirements when used as a reagent in
methods of detection. It is therefore appropriate to also
HAS ADOPTED THIS REGULATION:
provide for identical requirements for the equivalent
detection methods, where relevant. Part C of Chapter II
Article 1
of Annex I to Regulation (EC) No 2075/2005 should
therefore be amended accordingly.
Annex I to Regulation (EC) No 2075/2005 is amended in

accordance with the Annex to this Regulation.
(3) In
addition,
new
apparatuses
for Trichinella testing using
Article 2
the digestion method equivalent to the reference method
started being produced by private companies. Following
This Regulation shall enter into force on the 20th day following
these developments, guidelines for the validation of new
its publication in the Official Journal of the European Union.
This Regulation shall be binding in its entirety and directly applicable in all Member States.
Done at Brussels, 3 November 2011.
For the Commission
The President
José Manuel BARROSO
( 1 ) OJ L 139, 30.4.2004, p. 206.
( 2 ) OJ L 338, 22.12.2005, p. 60.
( 3 ) OJ L 281, 25.10.2007, p. 19.
( 4 ) http://www.iss.it/crlp/index.php

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L 287/24
EN
Official Journal of the European Union
4.11.2011
ANNEX
Chapter II of Annex I to Regulation (EC) No 2075/2005 is amended as follows:
1. in Part C, point 1(f) is replaced by the following:
‘(f) Pepsin, strength 1: 10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and
to 2 000 FIP (International Pharmaceutical Federation), or stabilised liquid pepsin with minimum 660 European
Pharmacopoeia units/ml’;
2. the following Part D is added:
‘D. Magnetic stirrer method for pooled sample digestion/‘on filter isolation’ and larva detection by a latex
agglutination test
This method is only considered equivalent for the testing of meat of domestic swine.
1. Apparatus and reagents
(a) Knife or scissors and tweezers for cutting specimens.
(b) Trays marked off into 50 squares, each of which can hold samples of approximately 2 g of meat, or other
tools giving equivalent guarantees as regards the traceability of the samples.
(c) A blender with a sharp chopping blade. Where the samples are larger than 3 g, a meat mincer with
openings of 2-4 mm or scissors must be used. In the case of frozen meat or tongue (after removal of the
superficial layer, which cannot be digested), a meat mincer is necessary and the sample size will need to be
increased considerably.
(d) Magnetic stirrers with thermostatically controlled heating plate and Teflon-coated stirring rods
approximately 5 cm long.
(e) Glass beakers, capacity 3 litres.
(f) Sieves,
mesh
size
180
microns,
external
diameter
11
cm,
with
stainless
steel
mesh.
(g) Steel filtration apparatus for 20 μm mesh filters with a steel funnel.
(h) Vacuum pump.
(i) Metal
or
plastic
tanks,
capacity
10-15
litres,
to
collect
the
digestive
juice.
(j) A
3D
gyratory
rocker.
(k) Aluminium foil.
(l) 25
%
hydrochloric
acid.
(m) Pepsin, strength: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Phar
macopoeia) and to 2 000 FIP (International Pharmaceutical Federation), or stabilised liquid pepsin with
minimum 660 European Pharmacopoeia units/ml.
(n) Tap water heated to 46-48 °C.
(o) A balance accurate to 0,1 g.
(p) Pipettes of different sizes (1, 10 and 25 ml), micropipettes according to the latex agglutination manu
facturer’s instructions and pipette holders.
(q) 20 microns nylon mesh filters of a diameter that fits with the filtration system.
(r) Plastic
or steel
forceps
of 10-15
cm.
(s) Conical vials of 15
ml.

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EN
4.11.2011 Official
Journal
of
the
European
Union L
287/25
(t) A
pestle
with
a
Teflon
or
steel
conical
tip
to
fit
in
the
conical
vials.
(u) A thermometer accurate to 0,5 °C within the range 1-100 °C.
(v) Latex agglutination cards of the Trichin-L antigen test kit validated under the code No
EURLP_D_001/2011.
(w) Buffer solution with preservative (sample diluent) of the Trichin-L antigen test kit validated under the code
No EURLP_D_001/2011.
(x) Buffer supplemented with preservative (negative control) of the Trichin-L antigen test kit validated under
the code No EURLP_D_001/2011.
(y) Buffer supplemented with Trichinella spiralis antigens and preservative (positive control) of the Trichin-L
antigen test kit validated under the code No EURLP_D_001/2011.
(z) Buffer with polystyrene particles coated with antibodies supplemented with preservative (latex beads) of
the Trichin-L antigen test kit validated under the code No EURLP_D_001/2011.
(aa) Disposable sticks.
2. Collecting of specimens
As stipulated in Chapter I(2).
3. Procedure
I. For complete pools (100 g of samples at a time), the procedure set out in points (a) to (i) of Chapter I(3)(I)
must be followed. In addition, the following procedure must be applied:
(a) The 20 microns nylon mesh filter is placed on the filtration support. The conical filtration steel funnel
is fixed to the support with the block system and the steel sieve of 180 microns mesh size is placed on
the funnel. The vacuum pump is connected with the filtration support and with the metal or plastic
tank, to collect the digestive fluid.
(b) Stop stirring and pour the digestion fluid into the filtration funnel through the sieve. Rinse the beaker
with 250 ml of warm water. The rinsing liquid must be poured into the filtration ramp after the
digested fluid has been successfully filtrated.
(c) With the forceps, take the filtration membrane holding it by an edge. Fold the filtration membrane in
four minimum and put it in the 15 ml conical tube.
(d) The filtration membrane is pushed at the bottom of the 15 ml conical tube with the help of the pestle
and strongly pressed by doing successive back and forth movements with the pestle which should be
positioned inside the filtration membrane folding according to the manufacturer’s instructions.
(e) The sample diluent is added into the 15 ml conical tube by pipette and the filtration membrane is
homogenised with the pestle by doing successive low amplitude back and forth movements, avoiding
abrupt movements to limit liquid splashes according to the manufacturer’s instructions.
(f) Each sample, the negative control, and the positive control, are dispensed into different fields of the
agglutination card by pipettes, according to the manufacturer’s instructions.
(g) The latex beads are added into each field of the agglutination card by a pipette, according to the
manufacturer’s instructions, without making them come into contact with the sample/s and controls. In
each field, the latex beads are then gently mixed with a disposable stick until the homogeneous liquid
covers the entire field.
(h) The agglutination card is put on the 3D rocker and is rocked according to the manufacturer’s
instructions.
(i) After the time established by the manufacturer’s instructions, the rocking is stopped and the aggluti
nation card is put on a plane surface and the reaction results are read. In the case of a positive sample,
the beads aggregates must appear. In the case of a negative sample, the suspension remains homo
geneous without beads aggregates.

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L 287/26
EN
Official Journal of the European Union
4.11.2011
(j) All equipment in contact with meat must be carefully decontaminated between runs by soaking for a
few seconds in warm water (60-90 °C). Surfaces upon which meat residues or inactivated larva/e remain
may be cleaned with a clean sponge and tap water. Once the procedure is finalised, a few drops of
detergent may be added to degrease the equipment. Then each piece must be thoroughly rinsed several
times in order to remove all traces of detergent.
(k) The pestle must be carefully decontaminated between runs by soaking for a few seconds in at least
250 ml of warm water (60-90 °C). Meat residues or inactivated larvae that could remain on its surface
must be eliminated with a clean sponge and tap water. Once the procedure is finalised, a few drops of
detergent may be added to degrease the pestle. Then the pestle must be thoroughly rinsed several times
in order to remove all traces of detergent.
II. Pools of less than 100 g as stipulated in Chapter I(3)(II)
For pools of less than 100 g, the procedure set out in Chapter I(3)(II) must be followed.
III. Positive or doubtful results
Where examination of a collective sample produces a positive or uncertain latex agglutination result, a
further 20 g sample is taken from each swine in accordance with Chapter I(2)(a). The 20 g samples from
five swine are pooled and examined using the method described in point I. In this way samples from 20
groups of five swine must be examined.
When a positive latex agglutination is obtained from a group of five swine, further 20 g samples are
collected from the individuals in the group and each is examined separately using one of the methods
described in Chapter I.
Parasite samples must be kept in 90 % ethyl alcohol for conservation and identification at species level at
the EU or national reference laboratory.
After parasite collection, positive fluids must be decontaminated by heating to at least 60 °C.’.

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